Aptamers with binding affinity to norovirus

ABSTRACT

The instant disclosure provides norovirus-binding aptamers, compositions comprising such aptamers, and methods of using and producing such aptamers. The aptamers are useful, for example, for detecting the presence of norovirus in test samples, for capturing and/or concentrating norovirus from test samples, for evaluating the efficacy of therapeutic agents in patients diagnosed with a norovirus infection, and for evaluating the efficacy of norovirus vaccines.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/317,002, which is a national stage filing under 35 U.S.C. 371 ofPCT/US2015/035619 filed Jun. 12, 2015, which International Applicationwas published by the International Bureau in English on Dec. 17, 2015,and application claims priority from U.S. Provisional Application No.62/011,880, filed Jun. 13, 2014, which applications are herebyincorporated in their entirety by reference in this application.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The present invention was made with United States Government supportunder grant number 2011-68003-30395 from USDA National Institute of Foodand Agriculture, Agriculture and Food Research Initiative. The UnitedStates Government has certain rights in this invention.

SEQUENCE LISTING

A sequence listing is incorporated herein by reference in its entirety.The listing, in ASCII format, was created on Apr. 9, 2019, is namedN61824_1450US_D1_-280_8_SEQLIST.txt and is 38.2 kilobytes in size.

BACKGROUND

Noroviruses are the most common cause of acute viral gastroenteritisworldwide (Glass et al., N. Engl. J. Med. 361:1776-1785 (2009)). Theseviruses, which are members of the Caliciviridae family, are transmittedby a variety of routes and frequently cause outbreaks in closed settingssuch as schools, nursing homes, and cruise ships. Noroviruses can alsobe transmitted through contaminated foods and water, and they are theleading cause of foodborne disease in the U.S. (Scallan et al., Emerg.Infect. Dis. 17:16-22 (2011)) and perhaps worldwide (Patel et al.,Emerg. Infect. Dis. 14:1224-1231 (2008); Ahmed et al., PLoS One 8:e75922(2013)). Low infectious dose, high virus concentrations in the feces andvomitus of infected individuals, lengthy environmental persistence, andresistance to many commonly used sanitizers and disinfectants allcontribute to the high degree of transmissibility of noroviruses (Hallet al., Emerg. Infect. Dis. 19:1198-1205 (2013)).

Despite their public health significance, routine detection ofnoroviruses in community settings or in food and environmental samplesis limited. First, there is no cell culture model to propagate theseviruses. Second, noroviruses have tremendous antigenic diversity. Thisantigenic diversity has complicated the development of broadly reactiveantibodies, meaning that enzyme immunoassays have poor sensitivity(Costantini et al., J. Clin. Microbiol. 48:2770-2778 (2010); Kele etal., Diagn. Microbiol. Infect. Dis. 70:475-478 (2011)). Detection ofnoroviruses in food and environmental samples is even more complicatedbecause virus concentrations are so low in these samples that it isnecessary to perform labor-intensive and inefficient pre-concentrationsteps prior to detection (Knight et al., Crit. Rev. Microbiol.39:295-309 (2013)).

SUMMARY OF THE CLAIMED INVENTION

Compositions are provided comprising isolated norovirus-bindingaptamers. Also provided are methods using norovirus-binding aptamers.Such methods include methods of detecting the presence of at least onenorovirus strain in a test sample, methods of capturing at least onenorovirus from a test sample, methods of evaluating the efficacy of atherapeutic agent in a patient diagnosed with a norovirus infection withat least one norovirus strain, and methods of evaluating the efficacy ofa norovirus vaccine. Also provided are mixtures comprising isolatednorovirus-binding aptamers and kits for detection of at least onenorovirus strain comprising isolated norovirus-binding aptamers.

The invention provides methods of detecting the presence of at least onenorovirus strain in a test sample, the methods comprising: (a)contacting said test sample with a norovirus-binding aptamer comprisinga norovirus-binding motif and/or any one of SEQ ID NOS: 1-78 or variantsthereof having at least 90% sequence identity and havingnorovirus-binding activity; and (b) detecting the presence of saidnorovirus-binding aptamer bound to norovirus in said test sample,wherein detection of bound aptamer indicates the presence of at leastone norovirus strain. The invention provides methods of detecting thepresence of at least one norovirus strain in a test sample, the methodscomprising: (a) contacting said test sample with a norovirus-bindingaptamer comprising a norovirus-binding motif and/or any one of SEQ IDNOS: 1-78 and 176-181 or variants thereof having at least 90% sequenceidentity and having norovirus-binding activity; and (b) detecting thepresence of said norovirus-binding aptamer bound to norovirus in saidtest sample, wherein detection of bound aptamer indicates the presenceof at least one norovirus strain. Optionally, the methods furthercomprise removing unbound norovirus-binding aptamer prior to detectingthe presence of said norovirus-binding aptamer bound to norovirus insaid test sample. Optionally, the detecting step comprises amplifyingthe bound aptamer.

Some methods further comprise comparing the presence of saidnorovirus-binding aptamer bound to norovirus in said test sample withthe presence of said norovirus-binding aptamer bound to norovirus in acontrol sample, whereby increased presence of said norovirus-bindingaptamer bound to norovirus in said test sample relative to said controlsample indicates the presence of at least one norovirus strain in saidtest sample. Optionally, said control sample and said test sample are ofthe same type.

In any of the above methods, the test sample can be a clinical samplesuch as a fecal sample. In any of the above methods, the test sample canbe an environmental sample. In any of the above methods, the test samplecan be a food sample.

In some methods, the test sample comprises captured norovirus.Optionally, said captured norovirus was captured through an initialcapturing step using a molecule that binds to said at least onenorovirus strain at an epitope that is different from the epitope towhich said norovirus-binding aptamer binds.

Some methods further comprise capturing said at least one norovirusstrain from said test sample by substantially separating theaptamer-bound norovirus from the remainder of said test sample.Optionally, the concentration of the captured norovirus is higher thanthe concentration of said at least one norovirus strain in said testsample.

In some methods, said norovirus-binding aptamer preferentially binds toinfectious norovirus particles. In some methods, said norovirus-bindingaptamer preferentially binds to norovirus strains from multiplegenogroups. In some methods, said norovirus-binding aptamerpreferentially binds to norovirus strains from genogroup I; genogroup I,genotype 1; genogroup II; genogroup II, genotype 2; and/or genogroup II,genotype 4. In some methods, said norovirus-binding aptamerpreferentially binds to an epitope within the VPg protein, the VP1protein, the P domain of the VP1 protein, the P1 subdomain of the VP1protein, the P2 subdomain of the VP1 protein, the S domain of the VP1protein, and/or the VP2 protein of said at least one norovirus strain.

In some methods, said norovirus-binding aptamer is a single-stranded DNAaptamer. In some methods, said norovirus-binding aptamer comprises anyone of the nucleic acid sequences of SMV-19 (SEQ ID NO: 25), SMV-21 (SEQID NO: 27), SMV-25 (SEQ ID NO: 30), SMV-26 (SEQ ID NO: 31), SMV-5 (S-7)(SEQ ID NO: 5), SMV-18 (SEQ ID NO: 6), SMV-22 (S-7) (SEQ ID NO: 7),SMV-5 (S-9) (SEQ ID NO: 12), SMV-17 (SEQ ID NO: 23), SMV-22 (S-9) (SEQID NO: 28), M1 (SEQ ID NO: 35), M6-2 (SEQ ID NO: 39), NV 1-1 (SEQ ID NO:41), NV 1-15 (SEQ ID NO: 42), NV 1-24 (SEQ ID NO: 53), NV 2-9 (SEQ IDNO: 55), NV 2-3 (SEQ ID NO: 56), NV 2-1 (SEQ ID NO:62), N6 (SEQ ID NO:66), N3 (SEQ ID NO: 67), N1 (SEQ ID NO: 68), N14 (SEQ ID NO: 69), N1-2(SEQ ID NO: 70), N4-2 (SEQ ID NO: 71), N11-12 (SEQ ID NO: 72), N12-2(SEQ ID NO: 73), T5 (SEQ ID NO: 74), T9 (SEQ ID NO: 75), T1-2 (SEQ IDNO: 76), T9-2 (SEQ ID NO: 77), and T10-2 (SEQ ID NO: 78). In somemethods, said norovirus-binding aptamer comprises any one of the nucleicacid sequences of SMV-19 (SEQ ID NO: 25), SMV-21 (SEQ ID NO: 27), SMV-25(SEQ ID NO: 30), SMV-26 (SEQ ID NO: 31), SMV-5 (S-7) (SEQ ID NO: 5),SMV-18 (SEQ ID NO: 6), SMV-22 (S-7) (SEQ ID NO: 7), SMV-5 (S-9) (SEQ IDNO: 12), SMV-17 (SEQ ID NO: 23), SMV-22 (S-9) (SEQ ID NO: 28), M1 (SEQID NO: 35), M9-2 (SEQ ID NO: 36), M12-2 (SEQ ID NO: 37), M13-2 (SEQ IDNO: 38), M6-2 (SEQ ID NO: 39), M5 (SEQ ID NO: 40), NV 1-1 (SEQ ID NO:41), NV 1-15 (SEQ ID NO: 42), NV 1-24 (SEQ ID NO: 53), NV 2-9 (SEQ IDNO: 55), NV 2-3 (SEQ ID NO: 56), NV 2-1 (SEQ ID NO:62), N6 (SEQ ID NO:66), N3 (SEQ ID NO: 67), N1 (SEQ ID NO: 68), N14 (SEQ ID NO: 69), N1-2(SEQ ID NO: 70), N4-2 (SEQ ID NO: 71), N11-12 (SEQ ID NO: 72), N12-2(SEQ ID NO: 73), T5 (SEQ ID NO: 74), T9 (SEQ ID NO: 75), T1-2 (SEQ IDNO: 76), T9-2 (SEQ ID NO: 77), T10-2 (SEQ ID NO: 78), AP1-GI (SEQ ID NO:176), AP2-GI (SEQ ID NO: 177), AP3-GI (SEQ ID NO: 178), AP4-GI (SEQ IDNO: 179), AP5-GI (SEQ ID NO: 180), and AP6-GI (SEQ ID NO: 181). In somemethods, said norovirus-binding motif comprises one or more of motifs1-14 or SEQ ID NOS: 98-145 and 162-174. In some methods, saidnorovirus-binding motif comprises one or more of motifs 1-23 or SEQ IDNOS: 98-145, 162-174, and 182-199.

In some methods, said norovirus-binding aptamer binds to said at leastone norovirus strain with an affinity characterized by a K_(d) valuefrom about 1 nM to about 999 nM. In some methods, said norovirus-bindingaptamer is at least 20 nucleotides in length. Optionally, saidnorovirus-binding aptamer is 20 to 80 nucleotides in length.

In some methods, said norovirus-binding aptamer is tethered to a solidsupport. Optionally, said norovirus-binding aptamer is immobilized on amagnetic bead.

In some methods, said norovirus-binding aptamer further comprises alabel. Optionally, said label is a fluorophore label or a biotin label.Optionally, said label is conjugated to the 5′ end of the aptamer.

In some methods, said norovirus-binding aptamer comprises an aptamermixture comprising a first aptamer and at least one different aptamer.Optionally, said first aptamer and said at least one different aptamerbind to different epitopes on the same norovirus strain. Optionally,said first aptamer and said at least one different aptamerpreferentially bind to different norovirus strains, wherein detection ofbound first aptamer and bound at least one different aptamer indicatesthe presence of at least two norovirus strains. Optionally, said firstaptamer and said at least one different aptamer are differentiallylabeled.

The invention also provides methods of capturing at least one norovirusfrom a test sample, the methods comprising: (a) contacting said testsample with a norovirus-binding aptamer comprising a norovirus-bindingmotif and/or any one of SEQ ID NOS: 1-78 or variants thereof having atleast 90% sequence identity and having norovirus-binding activity; and(b) substantially separating the aptamer-bound norovirus from theremainder of said test sample. The invention also provides methods ofcapturing at least one norovirus from a test sample, the methodscomprising: (a) contacting said test sample with a norovirus-bindingaptamer comprising a norovirus-binding motif and/or any one of SEQ IDNOS: 1-78 and 176-181 or variants thereof having at least 90% sequenceidentity and having norovirus-binding activity; and (b) substantiallyseparating the aptamer-bound norovirus from the remainder of said testsample. Optionally, the concentration of the captured norovirus ishigher than the concentration of said at least one norovirus in saidtest sample. Optionally, said norovirus-binding motif comprises one ormore of motifs 1-14 or SEQ ID NOS: 98-145 and 162-174. Optionally, saidnorovirus-binding motif comprises one or more of motifs 1-23 or SEQ IDNOS: 98-145, 162-174, and 182-199. Optionally, said norovirus-bindingaptamer binds to said at least one norovirus with an affinitycharacterized by a K_(d) value from about 1 nM to about 999 nM.

The invention also provides compositions comprising an isolated aptamercomprising a norovirus-binding motif and/or any one of SEQ ID NOS: 1-78or variants thereof having at least 90% sequence identity and havingnorovirus-binding activity, wherein said aptamer binds to at least onenorovirus strain. The invention also provides compositions comprising anisolated aptamer comprising a norovirus-binding motif and/or any one ofSEQ ID NOS: 1-78 and 176-181 or variants thereof having at least 90%sequence identity and having norovirus-binding activity, wherein saidaptamer binds to at least one norovirus strain. Optionally, saidisolated aptamer is a single-stranded DNA aptamer.

In some compositions, said isolated aptamer preferentially binds tonorovirus strains from multiple genogroups. In some compositions, saidisolated aptamer preferentially binds to norovirus strains fromgenogroup I; genogroup I, genotype 1; genogroup II; genogroup II,genotype 2; and/or genogroup II, genotype 4. In some compositions,binding of said isolated aptamer to said at least one norovirus strainis greater than binding of said isolated aptamer to hepatitis A virusand/or poliovirus. In some compositions, said isolated aptamerpreferentially binds to infectious norovirus particles. In somecompositions, said isolated aptamer preferentially binds to an epitopewithin the VPg protein, the VP1 protein, the P domain of the VP1protein, the P1 subdomain of the VP1 protein, the P2 subdomain of theVP1 protein, the S domain of the VP1 protein, and/or the VP2 protein ofsaid at least one norovirus strain.

In some compositions, said isolated aptamer comprises any one of thenucleic acid sequences of SMV-19 (SEQ ID NO: 25), SMV-21 (SEQ ID NO:27), SMV-25 (SEQ ID NO: 30), SMV-26 (SEQ ID NO: 31), SMV-5 (S-7) (SEQ IDNO: 5), SMV-18 (SEQ ID NO: 6), SMV-22 (S-7) (SEQ ID NO: 7), SMV-5 (S-9)(SEQ ID NO: 12), SMV-17 (SEQ ID NO: 23), SMV-22 (S-9) (SEQ ID NO: 28),M1 (SEQ ID NO: 35), M6-2 (SEQ ID NO: 39), NV 1-1 (SEQ ID NO: 41), NV1-15 (SEQ ID NO: 42), NV 1-24 (SEQ ID NO: 53), NV 2-9 (SEQ ID NO: 55),NV 2-3 (SEQ ID NO: 56), NV 2-1 (SEQ ID NO:62), N6 (SEQ ID NO: 66), N3(SEQ ID NO: 67), N1 (SEQ ID NO: 68), N14 (SEQ ID NO: 69), N1-2 (SEQ IDNO: 70), N4-2 (SEQ ID NO: 71), N11-12 (SEQ ID NO: 72), N12-2 (SEQ ID NO:73), T5 (SEQ ID NO: 74), T9 (SEQ ID NO: 75), T1-2 (SEQ ID NO: 76), T9-2(SEQ ID NO: 77), and T10-2 (SEQ ID NO: 78). In some compositions, saidisolated aptamer comprises any one of the nucleic acid sequences ofSMV-19 (SEQ ID NO: 25), SMV-21 (SEQ ID NO: 27), SMV-25 (SEQ ID NO: 30),SMV-26 (SEQ ID NO: 31), SMV-5 (S-7) (SEQ ID NO: 5), SMV-18 (SEQ ID NO:6), SMV-22 (S-7) (SEQ ID NO: 7), SMV-5 (S-9) (SEQ ID NO: 12), SMV-17(SEQ ID NO: 23), SMV-22 (S-9) (SEQ ID NO: 28), M1 (SEQ ID NO: 35), M9-2(SEQ ID NO: 36), M12-2 (SEQ ID NO: 37), M13-2 (SEQ ID NO: 38), M6-2 (SEQID NO: 39), M5 (SEQ ID NO: 40), NV 1-1 (SEQ ID NO: 41), NV 1-15 (SEQ IDNO: 42), NV 1-24 (SEQ ID NO: 53), NV 2-9 (SEQ ID NO: 55), NV 2-3 (SEQ IDNO: 56), NV 2-1 (SEQ ID NO:62), N6 (SEQ ID NO: 66), N3 (SEQ ID NO: 67),N1 (SEQ ID NO: 68), N14 (SEQ ID NO: 69), N1-2 (SEQ ID NO: 70), N4-2 (SEQID NO: 71), N11-12 (SEQ ID NO: 72), N12-2 (SEQ ID NO: 73), T5 (SEQ IDNO: 74), T9 (SEQ ID NO: 75), T1-2 (SEQ ID NO: 76), T9-2 (SEQ ID NO: 77),T10-2 (SEQ ID NO: 78), AP1-GI (SEQ ID NO: 176), AP2-GI (SEQ ID NO: 177),AP3-GI (SEQ ID NO: 178), AP4-GI (SEQ ID NO: 179), AP5-GI (SEQ ID NO:180), and AP6-GI (SEQ ID NO: 181). In some compositions, saidnorovirus-binding motif comprises one or more of motifs 1-14 or SEQ IDNOS: 98-145 and 162-174. In some compositions, said norovirus-bindingmotif comprises one or more of motifs 1-23 or SEQ ID NOS: 98-145,162-174, and 182-199.

In some compositions, the binding of said isolated aptamer to said atleast one norovirus strain is characterized by a K_(d) value from about1 nM to about 999 nM. In some compositions, said isolated aptamer is atleast 20 nucleotides in length. Optionally, said isolated aptamer is 20to 80 nucleotides in length.

In some compositions, said isolated aptamer is tethered to a solidsupport. Optionally, wherein said isolated aptamer is immobilized on amagnetic bead. In some compositions, said isolated aptamer furthercomprises a label. Optionally, said label is a fluorophore label or abiotin label. Optionally, said label is conjugated to the 5′ end of theaptamer.

The invention also provides aptamer mixtures comprising a first isolatedaptamer as described above and at least one different isolated aptameror aptamer mixtures comprising a first isolated aptamer and at least onedifferent isolated aptamer, wherein said first isolated aptamer and saidat least one different isolated aptamer are each isolated aptamers asdescribed above. Optionally, said first isolated aptamer and said atleast one different isolated aptamer bind to different epitopes on thesame norovirus strain. Optionally, said first isolated aptamer and saidat least one different isolated aptamer preferentially binds todifferent norovirus strains. Optionally, said at least one differentisolated aptamer preferentially binds to a target other than anorovirus. Optionally, said first isolated aptamer and said at least onedifferent isolated aptamer are differentially labeled.

The invention also provides kits for detection of at least one norovirusstrain comprising an isolated aptamer as described above and writtenmaterial describing a method for the kit's use. Optionally, the kitsfurther comprise a positive control comprising a norovirusvirus-like-particle suspension, viral RNA, or a surrogate virus.Optionally, the kits further comprise magnetic beads. Optionally, thekits further comprise primers and probes targeting a region of thegenome of at least one norovirus strain.

The invention also provides methods of evaluating the efficacy of atherapeutic agent in a patient diagnosed with a norovirus infection withat least one norovirus strain, the methods comprising: (a) contacting afirst clinical sample from said patient, obtained prior to treatmentwith said therapeutic agent, with a norovirus-binding aptamer comprisinga norovirus-binding motif and/or any one of SEQ ID NOS: 1-78 or variantsthereof having at least 90% sequence identity and havingnorovirus-binding activity; (b) detecting the presence of saidnorovirus-binding aptamer bound to norovirus in said first clinicalsample; (c) contacting a second clinical sample from said patient,obtained following treatment with said therapeutic agent, with saidnorovirus-binding aptamer; (d) detecting the presence of saidnorovirus-binding aptamer bound to norovirus in said second clinicalsample; and (e) comparing the presence of said norovirus-binding aptamerbound to norovirus in said first clinical sample with the presence ofsaid norovirus-binding aptamer bound to norovirus in said secondclinical sample, whereby decreased presence of said norovirus-bindingaptamer bound to norovirus in said second clinical sample relative tosaid first clinical sample indicates that said therapeutic agent iseffective in treating said norovirus infection in said patient. Theinvention also provides methods of evaluating the efficacy of atherapeutic agent in a patient diagnosed with a norovirus infection withat least one norovirus strain, the methods comprising: (a) contacting afirst clinical sample from said patient, obtained prior to treatmentwith said therapeutic agent, with a norovirus-binding aptamer comprisinga norovirus-binding motif and/or any one of SEQ ID NOS: 1-78 and 176-181or variants thereof having at least 90% sequence identity and havingnorovirus-binding activity; (b) detecting the presence of saidnorovirus-binding aptamer bound to norovirus in said first clinicalsample; (c) contacting a second clinical sample from said patient,obtained following treatment with said therapeutic agent, with saidnorovirus-binding aptamer; (d) detecting the presence of saidnorovirus-binding aptamer bound to norovirus in said second clinicalsample; and (e) comparing the presence of said norovirus-binding aptamerbound to norovirus in said first clinical sample with the presence ofsaid norovirus-binding aptamer bound to norovirus in said secondclinical sample, whereby decreased presence of said norovirus-bindingaptamer bound to norovirus in said second clinical sample relative tosaid first clinical sample indicates that said therapeutic agent iseffective in treating said norovirus infection in said patient.Optionally, the methods further comprise administering said therapeuticagent to said patient. Optionally, said norovirus-binding motifcomprises one or more of motifs 1-14 or SEQ ID NOS: 98-145 and 162-174.Optionally, said norovirus-binding motif comprises one or more of motifs1-23 or SEQ ID NOS: 98-145, 162-174, and 182-199. Optionally, saidnorovirus-binding aptamer comprises an aptamer mixture comprising afirst aptamer and at least one different aptamer. Optionally, said firstaptamer and said at least one different aptamer preferentially bind todifferent norovirus strains. Optionally, said first aptamer and said atleast one different aptamer are differentially labeled.

The invention also provides methods of evaluating the efficacy of anorovirus vaccine, the methods comprising: (a) challenging one or morevaccinated subjects and one or more non-vaccinated subjects withnorovirus; (b) contacting a first set of one or more clinical samplesfrom said one or more vaccinated subjects with a norovirus-bindingaptamer comprising a norovirus-binding motif and/or any one of SEQ IDNOS: 1-78 or variants thereof having at least 90% sequence identity andhaving norovirus-binding activity; (c) detecting the presence of saidnorovirus-binding aptamer bound to norovirus in said first set of one ormore clinical samples; (d) contacting a second set of one or moreclinical samples from said one or more non-vaccinated subjects with saidnorovirus-binding aptamer; (e) detecting the presence of saidnorovirus-binding aptamer bound to norovirus in said second set of oneor more clinical samples; and (f) comparing the presence of saidnorovirus-binding aptamer bound to norovirus in said first set of one ormore clinical samples with the presence of said norovirus-bindingaptamer bound to norovirus in said second set of one or more clinicalsamples, whereby lower presence of said norovirus-binding aptamer boundto norovirus in said first set relative to said second set indicatesthat said norovirus vaccine is effective in preventing or reducingnorovirus infection. The invention also provides methods of evaluatingthe efficacy of a norovirus vaccine, the methods comprising: (a)challenging one or more vaccinated subjects and one or morenon-vaccinated subjects with norovirus; (b) contacting a first set ofone or more clinical samples from said one or more vaccinated subjectswith a norovirus-binding aptamer comprising a norovirus-binding motifand/or any one of SEQ ID NOS: 1-78 and 176-181 or variants thereofhaving at least 90% sequence identity and having norovirus-bindingactivity; (c) detecting the presence of said norovirus-binding aptamerbound to norovirus in said first set of one or more clinical samples;(d) contacting a second set of one or more clinical samples from saidone or more non-vaccinated subjects with said norovirus-binding aptamer;(e) detecting the presence of said norovirus-binding aptamer bound tonorovirus in said second set of one or more clinical samples; and (f)comparing the presence of said norovirus-binding aptamer bound tonorovirus in said first set of one or more clinical samples with thepresence of said norovirus-binding aptamer bound to norovirus in saidsecond set of one or more clinical samples, whereby lower presence ofsaid norovirus-binding aptamer bound to norovirus in said first setrelative to said second set indicates that said norovirus vaccine iseffective in preventing or reducing norovirus infection. Optionally,said norovirus-binding motif comprises one or more of motifs 1-14 or SEQID NOS: 98-145 and 162-174. Optionally, said norovirus-binding motifcomprises one or more of motifs 1-23 or SEQ ID NOS: 98-145, 162-174, and182-199. Optionally, said norovirus-binding aptamer comprises an aptamermixture comprising a first aptamer and at least one different aptamer.Optionally, said first aptamer and said at least one different aptamerpreferentially bind to different norovirus strains. Optionally, saidfirst aptamer and said at least one different aptamer are differentiallylabeled.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the predicted structural folding of aptamer SMV-19 (SEQ IDNO: 79).

FIG. 2 shows the predicted structural folding of aptamer SMV-21 (SEQ IDNO: 80).

FIG. 3 shows the predicted structural folding of aptamer SMV-25 (SEQ IDNO: 81).

FIG. 4 shows the predicted structural folding of aptamer SMV-26 (SEQ IDNO: 82).

FIG. 5A-D shows equilibrium dissociation curves for aptamers SMV-19(FIG. 5A), SMV-21 (FIG. 5B), SMV-25 (FIG. 5C), and SMV-26 (FIG. 5D),respectively, with SMV VLPs as demonstrated by ELASA.

FIG. 6 shows binding of aptamer SMV-25 to HuNoV stool specimens asdemonstrated by ELASA using PBS and HuNoV-negative human stoolsuspensions (NVF) as negative controls. Statistically significantdifferences between the positive stool specimens and the HuNoV-negativehuman stool suspensions are designated with an asterisk (p<0.05)

FIG. 7 shows the performance of the AMC-RT-qPCR method using aptamerSMV-25 applied to lettuce samples artificially inoculated with HuNoVGII.4 fecal stock. Statistically significant differences between testsamples and negative controls (virus capture done using blocked beadswithout aptamer) are designated with an asterisk (p<0.05).

FIG. 8 shows binding of aptamers SMV-19, SMV-21, SMV-25, and SMV-26 tohepatitis A virus (HAV), poliovirus, and SMV VLPs as demonstrated byELASA. Statistically significant differences between the positivecontrol (SMV) and HAV or poliovirus are designated with an asterisk(p<0.05).

FIG. 9 shows the performance (% capture efficiency, CE) of theAMC-RT-qPCR method as applied to SMV using aptamer SMV-17 as the captureligand. The performance of immunomagnetic separation (IMS)-RT-qPCR wasperformed in tandem to provide comparison.

FIG. 10 shows the performance of the two-site binding sandwich qPCRmethod as applied to serially diluted SMV using amplification of aptamerSMV-22 (S-9) to achieve detection. An asterisk designates statisticalsignificance (p<0.05) when comparing the Ct values of samples containingSMV to that of the negative control which did not contain SMV.

FIG. 11 shows the performance of the IMS-RT-qPCR method as applied toserially diluted SMV. An asterisk designates statistical significance(p<0.05) when comparing the Ct values of samples containing SMV to thatof the negative control which did not contain SMV.

FIG. 12 shows the predicted structural folding of aptamer M1 (SEQ ID NO:83).

FIG. 13 shows the predicted structural folding of aptamer M6-2 (SEQ IDNO: 84).

FIG. 14 shows the predicted structural folding of aptamer M5 (SEQ ID NO:97).

FIG. 15 shows binding ratios of partially purified, serially diluted 20%GII.4 and human NoV-negative stool samples to selected aptamers byELASA. Data are presented as ratio of absorbance for test samples(serially diluted stool) vs. PBS negative control wells. The asteriskindicates a statistically significant difference (p<0.05) between GII.4positive stool and stool confirmed negative for human NoV. Error barsrepresent one standard deviation above/below the mean. X axis is labeledby aptamer designation and dilution of stool.

FIG. 16 shows capture and subsequent detection of GII.4 New Orleans fromhuman stool using combined AMC-RT-qPCR. Negative controls consisted ofbeads without the aptamer conjugates. An asterisk designates statisticalsignificance (p<0.05) when comparing aptamer and no aptamer controls.

FIG. 17 shows binding of aptamer M6-2 and HBGA Type A to GII.4 HoustonVirus VLPs at different temperatures.

FIG. 18A-B shows binding of aptamer M6-2 and HBGA Type A to GII.4Houston Virus VLPs at 63° C. (FIG. 18A) and 65° C. (FIG. 18B) fordifferent times.

FIG. 19 shows a binding comparison of two different human NoV aptamersto SMV VLPs treated at different temperatures for one minute as apercentage of untreated SMV VLPs. Percentages have absorbance ofcompletely denatured SMV subtracted.

FIG. 20A-B shows degradation of SMV VLPs at two selected temperatures.

FIG. 20A shows signal of SMV VLPs treated at 72° C. for different timesas a percentage of untreated SMV VLPs. FIG. 20B shows signal of SMV VLPsat 70° C. for different times as a percentage of untreated SMV VLPs.Percentages have absorbance of completely denatured SMV subtracted.

FIG. 21A-B shows binding behaviour of three different ligands (aptamerM6-2, HBGA type A, and antibody NS14) to GII.4 Sydney Virus VLPs treatedat selected temperatures for different times. FIG. 21A shows bindingsignals to GII.4 Sydney Virus VLPs treated at 68° C. for different timesas a percentage of untreated GII.4 Sydney Virus VLPs. FIG. 21B showsbinding signals to GII.4 Sydney Virus VLPs treated at 65° C. fordifferent times as a percentage of untreated GII.4 Sydney Virus VLPs.Percentages have absorbance of completely denatured GII.4 Sydney Virussubtracted.

FIG. 22 shows binding signal of three human NoV ligands (aptamer M6-2,HBGA type A, and antibody NS14) to completely denatured GII.4 SydneyVirus VLPs as a percentage of untreated GII.4 Sydney Virus VLPs.Background signal as measured by no VLP wells was subtracted from allabsorbances.

FIG. 23 shows the predicted structural folding of aptamer Apt-GI (SEQ IDNO: 176).

FIG. 24 shows the predicted structural folding of aptamer Ap2-GI (SEQ IDNO: 177).

FIG. 25 shows the predicted structural folding of aptamer Ap3-GI (SEQ IDNO: 178).

FIG. 26 shows the predicted structural folding of aptamer Ap4-GI (SEQ IDNO: 179).

FIG. 27 shows the predicted structural folding of aptamer Ap5-GI (SEQ IDNO: 180).

FIG. 28 shows the predicted structural folding of aptamer Ap6-GI (SEQ IDNO: 181).

DETAILED DESCRIPTION I. Overview

The instant disclosure provides norovirus-binding aptamers, isolatednorovirus-binding aptamers, compositions comprising such aptamers, andmethods for using and producing such aptamers. The norovirus-bindingaptamers disclosed herein are useful, for example, for detecting thepresence of norovirus in test samples such as clinical samples,environmental samples, and food samples, and for capturing and/orconcentrating norovirus from test samples.

II. Aptamers

The ability of nucleic acids, and single-stranded nucleic acids inparticular, to fold into specific and stable secondary structures hasled to identification of nucleic acid sequences (i.e., aptamers) withstructures that can bind preferentially to selected targets and alsodiscriminate between subtle molecular differences within the target.

Unless otherwise apparent from context, the term “aptamer” refers to anucleic acid molecule that naturally folds into specific and stablesecondary structures that enable it to bind to a selected target. Theterm “nucleic acid” refers to single-stranded or double-strandeddeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and any chemicalmodifications thereof. Such modifications can include, for example,modifications at cytosine exocyclic amines, substitution of5-bromo-uracil, backbone modifications, methylations, unusualbase-pairing combinations, and the like. In some cases, aptamers areisolated nucleic acids.

The term “isolated,” when referring to an aptamer or a nucleic acid,means that the aptamer or nucleic acid is a predominant aptamer ornucleic acid species in a composition. An aptamer or nucleic acid can beconsidered to be a predominant aptamer or nucleic acid species in acomposition if it represents at least about 5%, at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95%, or 100% of the aptamers or nucleicacid species in the composition. This can be determined, for example,using analytical chemistry techniques such as polyacrylamide gelelectrophoresis, high performance liquid chromatography, and the like.

The term “norovirus-binding aptamer” refers to any aptamer thatpreferentially binds to any norovirus (i.e., has norovirus-bindingactivity). The term “preferential binding” or “preferentially binds”means that an aptamer or other molecule binds with greater affinity,with greater avidity, more readily, and/or with greater duration to atarget than it binds to at least one unrelated non-target. An aptamer orother molecule that preferentially binds to a first target may or maynot preferentially bind to a second target. Thus, preferential bindingdoes not require (although it can include) exclusive binding. Manyassays can be used to qualitatively or quantitatively detect or measurebinding of norovirus-binding aptamers to norovirus. For example, anEnzyme-Linked Aptamer Sorbent Assay (ELASA) can be used. Assaysinvolving amplification of the bound aptamer (e.g., qPCR) or RNA fromthe aptamer-bound virus (e.g., qRT-PCR) can be used. Flow cytometrymethods as described in U.S. Pat. No. 5,853,984 can be used.Microarrays, BIAcore assays, differential centrifugation,chromatography, electrophoresis, immunoprecipitation, opticalbiosensors, and other surface plasmon resonance assays can be used asdescribed in WO 2011/061351. Other assays that can be used arecalorimetric analysis and dot blot assays. Moreover, just as theenzyme-linked immunosorbent assay (ELISA) was adapted for aptamers inthe ELASA assay, any other assays involving norovirus-binding antibodiescan be adapted for use with the norovirus-binding aptamers disclosedherein in place of the antibodies. Such assays include immunometricassays such as radioimmunoassays, flow cytometry assays, blottingapplications, anisotropy, membrane assays, biosensors, and the like. Anyother assays known in the art can also be used or adapted to detect ormeasure binding of norovirus-binding aptamers to norovirus.

A. Structure and Examples of Norovirus-Binding Aptamers

The norovirus-binding aptamers disclosed herein can have discretenucleic acid structures that facilitate preferential binding tonorovirus targets. The primary sequence of a DNA is a specific string ofnucleotides (A, C, G, or T) in one dimension. Likewise, the primarysequence of an RNA is a specific string of nucleotides (A, C, G, or U)in one dimension. The primary sequence dictates the three dimensionalconfiguration of the aptamer. In some cases, the primary sequence of anorovirus-binding aptamer can be greater than 20 nucleotides.Optionally, it can be between about 20-200 nucleotides, between about20-150 nucleotides, between about 20-100 nucleotides, between about20-80 nucleotides, between about 20-50 nucleotides, between about 30-50nucleotides, or between about 35-50 nucleotides. Representative primarysequences for the norovirus-binding aptamers disclosed herein includeSEQ ID NOS: 1-78. Variants of SEQ ID NOS: 1-78 that retainnorovirus-binding activity can include aptamers comprising a nucleicacid sequence having at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, or 100% sequence identity with any one of SEQ IDNOS: 1-78. Other representative primary sequences for thenorovirus-binding aptamers disclosed herein include SEQ ID NOS: 176-181.Variants of SEQ ID NOS: 176-181 that retain norovirus-binding activitycan include aptamers comprising a nucleic acid sequence having at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or 100%sequence identity with any one of SEQ ID NOS: 176-181.

The secondary structure of a section of a DNA or RNA is represented bycontact in two dimensions between specific nucleotides. Secondarystructures can comprise Watson/Crick base pairs (A:U, A:T and C:G) andother base pairs of lower stability (e.g., G:U, G:T, A:C, G:A, U:U, andT:T). Secondary structures include stem loops, symmetric and asymmetricbulges, pseudoknots, and combinations of the same. In some cases, suchstructures can be formed in a nucleic acid sequence of no more thanabout 30 nucleotides. When nucleotides that are distant in the primarysequence and not thought to interact through Watson/Crick andnon-Watson/Crick base pairs are in fact interacting, these interactions(which are often depicted in two dimensions) are also part of thesecondary structure.

The tertiary structure of a DNA or RNA is the description in space ofthe atoms of the DNA or RNA. Primary sequences of aptamers limit thepossible tertiary structures, as do the fixed secondary structures.Norovirus-binding aptamers have structures in three dimensions that arecomprised of a collection of DNA or RNA motifs and secondary structures.DNA or RNA secondary and tertiary structures include all the ways inwhich it is possible to describe in general terms the most stable groupsof conformations that a nucleic acid compound can form.

Families of norovirus-binding aptamers that bind to a particularnorovirus target can be characterized by one or more norovirus-bindingmotifs held in common. Although such families of aptamers havingnorovirus-binding motifs in common may include a relatively large numberof potential members, the family members are capable of preferentialbinding to one or more particular norovirus targets.

The term “norovirus-binding motif” refers to any motif that is within anaptamer and is predicted to contribute to preferential binding of theaptamer to a norovirus. Motifs can be primary sequences of nucleotides,secondary structures or portions thereof, or tertiary structures orportions thereof. Examples of norovirus-binding motifs include motifs1-14 and SEQ ID NOS: 98-145 and 162-174 as disclosed herein. Otherexamples of norovirus-binding motifs include motifs 15-23 and SEQ IDNOS: 182-199 as disclosed herein. Yet other examples ofnorovirus-binding motifs include any nucleic acid sequences contributingto any of the secondary structures (e.g., stem loops, symmetric andasymmetric bulges, pseudoknots, and the like) in a norovirus-bindingaptamer, such as those shown in FIGS. 1-4, 12-14, and 23-28. Otherexamples of norovirus-binding motifs include nucleic acid sequences ofat least about 3, at least about 4, at least about 5, at least about 6,at least about 7, at least about 8, at least about 9, at least about 10nucleotides, at least about 11 nucleotides, at least about 12nucleotides, at least about 13 nucleotides, at least about 14nucleotides, at least about 15 nucleotides, at least about 16nucleotides, at least about 17 nucleotides, at least about 18nucleotides, at least about 19 nucleotides, or at least about 20nucleotides that share at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, or 100% sequence identity with each otherand are present in two or more norovirus-binding aptamers.

When comparing aptamer sequences, percentage sequence identities aredetermined with aptamer sequences maximally aligned. After alignment, ifa subject aptamer region (e.g., a putative norovirus-binding motif) isbeing compared with the same region of a reference aptamer, thepercentage sequence identity between the subject and reference aptamerregions is the number of positions occupied by the same nucleotide inboth the subject and reference aptamer regions divided by the totalnumber of aligned positions of the two regions, with gaps not counted,multiplied by 100 to convert to percentage.

One family of norovirus-binding aptamers comprises motif 1. Motif 1 ischaracterized by the sequence T-G-N₁-N₂-A-G-N₃-N₄ (SEQ ID NO: 98), whereN₁, N₂, N₃, and N₄ can be any nucleotide. Optionally, one or more of thenucleotides in the motif can be modified. In some cases, N₁ can be T orA. In some cases, N₂ can be T or G. In some cases, N₃ can be G, C, or A.In some cases, N₄ can be G or C. In some cases, N₁ can be T or A, N₂ canbe T or G, N₃ can be G, C, or A, N₄ can be G or C, and the motif has thesequence of T-G-W-K-A-G-V-S(SEQ ID NO: 162) using IUPAC nomenclature,where W can be A or T, K can be G or T, V can be A or G or C, and S canbe G or C. In one example of motif 1, N₁ is A, N₂ is T, N₃ is A, and N₄is G, and the motif has the sequence of T-G-A-T-A-G-A-G (SEQ ID NO:112). In another example of motif 1, N₁ is T, N₂ is T, N₃ is G, and N₄is G, and the motif has the sequence of T-G-T-T-A-G-G-G (SEQ ID NO:113). In yet another example of motif 1, N₁ is T, N₂ is T, N₃ is G, andN₄ is C, and the motif has the sequence of T-G-T-T-A-G-G-C(SEQ ID NO:114). Examples of norovirus-binding aptamers comprising motif 1 includeSMV-19 (SEQ ID NO: 25), SMV-21 (SEQ ID NO: 27), and SMV-26 (SEQ ID NO:31).

Another family of norovirus-binding aptamers comprises motif 2. Motif 2is characterized by the sequence N₁-N₂-N₃-T-G-T-N₄-N₅-N₆-G (SEQ ID NO:99), where N₁, N₂, N₃, N₄, N₅, and N₆ can be any nucleotide. Optionally,one or more nucleotides in the motif can be modified. In some cases, N₁can be C or G. In some cases, N₂ can be A, T, or C. In some cases, N₃can be G or A. In some cases, N₄ can be G, T, or C. In some cases, N₅can be T or A. In some cases, N₆ can be T, G, or A. In some cases, N₁can be C or G, N₂ can be A, T, or C, N₃ can be G or A, N₄ can be G, T,or C, N₅ can be T or A, N₆ can be T, G, or A, and the motif has thesequence of S-H-R-T-G-T-B-W-D-G (SEQ ID NO: 163) using IUPACnomenclature, where S can be G or C, H can be A or C or T, R can be G orA, B can be G or C or T, W can be A or T, and D can be A or G or T. Inone example of motif 2, N₁ is C, N₂ is A, N₃ is G, N₄ is G, N₅ is T, andN₆ is T, and the motif has the sequence of C-A-G-T-G-T-G-T-T-G (SEQ IDNO: 115). In another example of motif 2, N₁ is C, N₂ is C, N₃ is A, N₄is T, N₅ is T, and N₆ is T, and the motif has the sequence ofC-C-A-T-G-T-T-T-T-G (SEQ ID NO: 116). In yet another example of motif 2,N₁ is C, N₂ is T, N₃ is G, N₄ is G, N₅ is A, and N₆ is A, and the motifhas the sequence of C-T-G-T-G-T-G-A-A-G (SEQ ID NO: 117). In yet anotherexample of motif 2, N₁ is G, N₂ is A, N₃ is G, N₄ is C, N₅ is T, and N₆is G, and the motif has the sequence of G-A-G-T-G-T-C-T-G-G (SEQ ID NO:118). Examples of norovirus-binding aptamers comprising motif 2 includeSMV-19 (SEQ ID NO: 25), SMV-21 (SEQ ID NO: 27), SMV-25 (SEQ ID NO: 30),and SMV-26 (SEQ ID NO: 31).

Another family of norovirus-binding aptamers comprises motif 3. Motif 3is characterized by the sequence A-G-G-T-N-T (SEQ ID NO: 100), where Ncan be any nucleotide. Optionally, one or more nucleotides in the motifcan be modified. In some cases, N can be T or G, and the motif has thesequence of A-G-G-T-K-T (SEQ ID NO: 164) using IUPAC nomenclature, whereK can be G or T. In one example of motif 3, N is T, and the motif hasthe sequence of A-G-G-T-T-T (SEQ ID NO: 119). In another example ofmotif 3, N is G, and the motif has the sequence of A-G-G-T-G-T (SEQ IDNO: 120). Examples of norovirus-binding aptamers comprising motif 3include SMV-19 (SEQ ID NO: 25) and SMV-21 (SEQ ID NO: 27).

Another family of norovirus-binding aptamers comprises motif 4. Motif 4is characterized by the sequence T-G-G-G-N-A (SEQ ID NO: 101), where Ncan be any nucleotide. Optionally, one or more nucleotides in the motifcan be modified. In some cases, N can be G or A, and the motif has thesequence of T-G-G-G-R-A (SEQ ID NO: 165) using IUPAC nomenclature, whereR can be G or A. In one example of motif 4, N is G, and the motif hasthe sequence of T-G-G-G-G-A (SEQ ID NO: 121). In another example ofmotif 4, N is A, and the motif has the sequence of T-G-G-G-A-A (SEQ IDNO: 122). Examples of norovirus-binding aptamers comprising motif 4include M1 (SEQ ID NO: 35) and M6-2 (SEQ ID NO: 39).

Another family of norovirus-binding aptamers comprises motif 5. Motif 5is characterized by the sequence G-N₁-G-A-N₂-A-A (SEQ ID NO: 102), whereN can be any nucleotide. Optionally, one or more nucleotides in themotif can be modified. In some cases, N₁ can be G or T. In some cases,N₂ can be T or C. In some cases, N₁ can be G or T, N₂ can be T or C, andthe motif has the sequence of G-K-G-A-Y-A-A (SEQ ID NO: 166) using IUPACnomenclature, where K can be G or T and Y can be T or C. In one exampleof motif 5, N₁ is G, N₂ is T, and the motif has the sequence ofG-G-G-A-T-A-A (SEQ ID NO: 123). In another example of motif 5, N₁ is T,N₂ is C, and the motif has the sequence of G-T-G-A-C-A-A (SEQ ID NO:124). Examples of norovirus-binding aptamers comprising motif 5 includeM1 (SEQ ID NO: 35) and M5 (SEQ ID NO: 40).

Another family of norovirus-binding aptamers comprises motif 6. Motif 6is characterized by the sequence T-G-G-G-N₁-N₂-G (SEQ ID NO: 103), whereN₁ and N₂ can be any nucleotide. Optionally, one or more nucleotides inthe motif can be modified. In some cases, N₁ can be G or A. In somecases, N₂ can be G or A. In some cases, N₁ can be G or A, N₂ can be G orA, and the motif has the sequence of T-G-G-G-R-R-G (SEQ ID NO: 167)using IUPAC nomenclature, where R can be G or A. In one example of motif6, N₁ is G, N₂ is G, and the motif has the sequence of T-G-G-G-G-G-G(SEQ ID NO: 125). In another example of motif 6, N₁ is A, N₂ is A, andthe motif has the sequence of T-G-G-G-A-A-G (SEQ ID NO: 126). Examplesof norovirus-binding aptamers comprising motif 6 include M5 (SEQ ID NO:40) and M6-2 (SEQ ID NO: 39).

Another family of norovirus-binding aptamers comprises motif 7. Motif 7is characterized by the sequence T-C-N₁-N₂-G-T-A (SEQ ID NO: 104), whereN₁ and N₂ can be any nucleotide. Optionally, one or more nucleotides inthe motif can be modified. In some cases, N₁ can be G or C. In somecases, N₂ can be T or G. In some cases, N₁ can be G or C, N₂ can be T orG, and the motif has the sequence of T-C-S-K-G-T-A (SEQ ID NO: 168)using IUPAC nomenclature, where S can be G or C and K can be G or T. Inone example of motif 7, N₁ is C, N₂ is T, and the motif has the sequenceof T-C-G-T-G-T-A (SEQ ID NO: 127). In another example of motif 7, N₁ isC, N₂ is G, and the motif has the sequence of T-C-C-G-G-T-A (SEQ ID NO:128). Examples of norovirus-binding aptamers comprising motif 7 includeM1 (SEQ ID NO: 35) and M6-2 (SEQ ID NO: 39).

Another family of norovirus-binding aptamers comprises motif 8. Motif 8is characterized by the sequence G-G-T-N-C-G-G-T (SEQ ID NO: 105), whereN can be any nucleotide. Optionally, one or more nucleotides in themotif can be modified. In some cases, N can be G or C, and the motif hasthe sequence of G-G-T-S-C-G-G-T (SEQ ID NO: 169), where S can be G or C.In one example of motif 8, N is G, and the motif has the sequence ofG-G-T-G-C-G-G-T (SEQ ID NO: 129). In another example of motif 8, N is C,and the motif has the sequence of G-G-T-C-C-G-G-T (SEQ ID NO: 130).Examples of norovirus-binding aptamers comprising motif 8 include M5(SEQ ID NO: 40) and M6-2 (SEQ ID NO: 39).

Another family of norovirus-binding aptamers comprises motif 9. Motif 9is characterized by the sequence T-A-A-A-N₁-G-N₂-A (SEQ ID NO: 106),where N₁ and N₂ can be any nucleotide. Optionally, one or morenucleotides in the motif can be modified. In some cases, N₁ can be C orT. In some cases, N₂ can be T or C. In some cases, N₁ can be C or T, N₂can be T or C, and the motif has the sequence of T-A-A-A-Y-G-Y-A (SEQ IDNO: 170) using IUPAC nomenclature, where Y can be T or C. In one exampleof motif 9, N₁ is C, N₂ is T and the motif has the sequence ofT-A-A-A-C-G-T-A (SEQ ID NO: 131). In another example of motif 9, N₁ isT, N₂ is C, and the motif has the sequence of T-A-A-A-T-G-C-A (SEQ IDNO: 132). Examples of norovirus-binding aptamers comprising motif 9include M1 (SEQ ID NO: 35) and M6-2 (SEQ ID NO: 39).

Another family of norovirus-binding aptamers comprises motif 10. Motif10 is characterized by the sequence T-G-T-T-N₁-N₂-N₃-G-G-G-N₄-A-T-N₅-A-A(SEQ ID NO: 107), where N₁, N₂, N₃, N₄, and N₅ can be any nucleotide.Optionally, one or more nucleotides in the motif can be modified. Insome cases, N₁ can be T or A. In some cases, N₂ can be A or G. In somecases, N₃ can be T or G. In some cases, N₄ can be G or A. In some cases,N₅ can be T or A. In some cases, N₁ can be T or A, N₂ can be A or G, N₃can be T or G, N₄ can be G or A, N₅ can be T or A, and the motif has thesequence of T-G-T-T-W-R-K-G-G-G-R-A-T-W-A-A (SEQ ID NO: 171) using IUPACnomenclature, where W can be A or T, R can be G or A, and K can be G orT. In one example of motif 10, N₁ is T, N₂ is A, N₃ is T, N₄ is G, andN₅ is A, and the motif has the sequence ofT-G-T-T-T-A-T-G-G-G-G-A-T-A-A-A (SEQ ID NO: 133). In another example ofmotif 10, N₁ is A, N₂ is A, N₃ is G, N₄ is A, and N₅ is T, and the motifhas the sequence of T-G-T-T-A-A-G-G-G-G-A-A-T-T-A-A (SEQ ID NO: 134). Inanother example, N₁ is A, N₂ is G, N₃ is G, N₄ is A, and N₅ is T, andthe motif has the sequence of T-G-T-T-A-G-G-G-G-G-A-A-T-T-A-A (SEQ IDNO: 135). Examples of norovirus-binding aptamers comprising motif 10include M1 (SEQ ID NO: 35), M9-2 (SEQ ID NO: 36), and M12-2 (SEQ ID NO:37).

Another family of norovirus-binding aptamers comprises motif 11. Motif11 is characterized by the sequenceT-A-A-T-N₁-C-G-T-N₂-T-A-C-T-A-A-T-C-A (SEQ ID NO: 108), where N₁ and N₂can be any nucleotide. Optionally, one or more nucleotides in the motifcan be modified. In some cases, N₁ can be T or C. In some cases, N₂ canbe G or C. In some cases, N₁ can be T or C, N₂ can be G or C, and themotif has the sequence of T-A-A-T-Y-C-G-T-S-T-A-C-T-A-A-T-C-A (SEQ IDNO: 172) using IUPAC nomenclature, where Y can be T or C and S can be Gor C. In one example of motif 11, N₁ is T and N₂ is G, and the motif hasthe sequence of T-A-A-T-T-C-G-T-G-T-A-C-T-A-A-T-C-A (SEQ ID NO: 136). Inanother example of motif 11, N₁ is C and N₂ is C, and the motif has thesequence of T-A-A-T-C-C-G-T-C-T-A-C-T-A-A-T-C-A (SEQ ID NO: 137).Examples of norovirus-binding aptamers comprising motif 11 include M1(SEQ ID NO: 35) and M9-2 (SEQ ID NO: 36).

Another family of norovirus-binding aptamers comprises motif 12. Motif12 is characterized by the sequence T-G-G-G-N₁-N₂-G-N₃-G-G-T-N₄-C-G-G-T(SEQ ID NO: 109), where N₁, N₂, N₃, and N₄ can be any nucleotide.Optionally, one or more of the nucleotides in the motif can be modified.In some cases, N₁ can be G or A. In some cases, N₂ can be G or A. Insome cases, N₃ can be T or A. In some cases, N₄ can be G or C. In somecases, N₁ can be G or A, N₂ can be G or A, N₃ can be T or A, N₄ can be Gor C, and the motif has the sequence of T-G-G-G-R-R-G-W-G-G-T-S-C-G-G-T(SEQ ID NO: 173) using IUPAC nomenclature, where R can be G or A, W canbe A or T, and S can be G or C. In one example of motif 12, N₁ is G, N₂is G, N₃ is T, and N₄ is G, and the motif has the sequence ofT-G-G-G-G-G-G-T-G-G-T-G-C-G-G-T (SEQ ID NO: 138). In another example ofmotif 12, N₁ is A, N₂ is A, N₃ is A, and N₄ is C, and the motif has thesequence of T-G-G-G-A-A-G-A-G-G-T-C-C-G-G-T (SEQ ID NO: 139). Examplesof norovirus-binding aptamers comprising motif 12 include M5 (SEQ ID NO:40), M6-2 (SEQ ID NO: 39), and M13-2 (SEQ ID NO: 38).

Another family of norovirus-binding aptamers comprises motif 13. Motif13 is characterized by the sequence T-G-G-G-R₁-R₂-K (SEQ ID NO: 110)using IUPAC nomenclature, where R can be A or G and K can be G or T.Optionally, one or more nucleotides in the motif can be modified. In oneexample of motif 13, R₁ is G, R₂ is A, K is T, and the motif has thesequence of T-G-G-G-G-A-T (SEQ ID NO: 140). In another example of motif13, R₁ is G, R₂ is G, K is G, and the motif has the sequence ofT-G-G-G-G-G-G (SEQ ID NO: 141). In another example of motif 13, R₁ is A,R₂ is A, K is G, and the motif has the sequence of T-G-G-G-A-A-G (SEQ IDNO: 142). Examples of norovirus-binding aptamers comprising motif 13include M1 (SEQ ID NO: 35), M5 (SEQ ID NO: 40), and M6-2 (SEQ ID NO:39).

Another family of norovirus-binding aptamers comprises motif 14. Motif14 is characterized by the sequenceT-G-G-G-R₁-R₂-K₁-W-R₃-R₄-Y₁-S-Y₂-R₅-K₂-Y₃ (SEQ ID NO: 111) using IUPACnomenclature, where R can be A or G, K can be G or T, W can be A or T, Ycan be C or T, and S can be G or C. In some cases, K₁ can be G, R₃ canbe G, R₄ can be G, Y₁ can be T, Y₂ can be C, R₅ can be G, K₂ can be G,and Y₃ can be T, and the motif has the sequence ofT-G-G-G-R₁-R₂-G-W-G-G-T-S-C-G-G-T (SEQ ID NO: 174) using IUPACnomenclature, where R can be G or A, W can be T or A, and S can be G orC. In one example of motif 14, R₁ is G, R₂ is A, K₁ is T, W is A, R₃ isA, R₄ is A, Y₁ is C, S is G, Y₂ is T, R₅ is A, K₂ is T, Y₃ is C, and themotif has the sequence T-G-G-G-G-A-T-A-A-A-C-G-T-A-T-C(SEQ ID NO: 143).In another example of motif 14, R₁ is G, R₂ is G, K₁ is G, W is T, R₃ isG, R₄ is G, Y₁ is T, S is G, Y₂ 1 S C, R₅ is G, K₂ is G, Y₃ is T, andthe motif has the sequence T-G-G-G-G-G-G-T-G-G-T-G-C-G-G-T (SEQ ID NO:144). In another example of motif 14, R₁ is A, R₂ is A, K₁ is G, W is A,R₃ is G, R₄ is G, Y₁ is T, S is C, Y₂ is C, R₅ is G, K₂ is G, Y₃ is T,and the motif has the sequence T-G-G-G-A-A-G-A-G-G-T-C-C-G-G-T (SEQ IDNO: 145). Examples of norovirus-binding aptamers comprising motif 14include M1 (SEQ ID NO: 35), M5 (SEQ ID NO: 40), M6-2 (SEQ ID NO: 39),and M13-2 (SEQ ID NO: 38).

Another family of norovirus-binding aptamers comprises motif 15. Motif15 is characterized by the sequence A-C-G-A-A-T-G (SEQ ID NO: 182).Optionally, one or more nucleotides in the motif can be modified.Examples of norovirus-binding aptamers comprising motif 15 includeAP2-GI (SEQ ID NO: 177) and AP3-GI (SEQ ID NO: 178).

Another family of norovirus-binding aptamers comprises motif 16. Motif16 is characterized by the sequence A-C-G-G-A-T (SEQ ID NO: 183).Optionally, one or more nucleotides in the motif can be modified.Examples of norovirus-binding aptamers comprising motif 16 includeAP3-GI (SEQ ID NO: 178).

Another family of norovirus-binding aptamers comprises motif 17. Motif17 is characterized by the sequence C-G-A-A-G-G-G-A-C(SEQ ID NO: 184).Optionally, one or more nucleotides in the motif can be modified.Examples of norovirus-binding aptamers comprising motif 17 includeAP2-GI (SEQ ID NO: 177) and AP4-GI (SEQ ID NO: 179).

Another family of norovirus-binding aptamers comprises motif 18. Motif18 is characterized by the sequence C-G-A-A-G-T-G-T-A-C(SEQ ID NO: 185).Optionally, one or more nucleotides in the motif can be modified.Examples of norovirus-binding aptamers comprising motif 18 includeAP4-GI (SEQ ID NO: 179).

Another family of norovirus-binding aptamers comprises motif 19. Motif19 is characterized by the sequence G-G-N-G-A-G-C-G (SEQ ID NO: 186),where N can be any nucleotide. Optionally, one or more nucleotides inthe motif can be modified. In some cases, N can be A or T, and the motifhas the sequence G-G-W-G-A-G-C-G (SEQ ID NO: 191) using IUPACnomenclature, where W can be A or T. In one example of motif 19, W is A,and the motif has the sequence of G-G-A-G-A-G-C-G (SEQ ID NO: 192). Inanother example of motif 19, W is T, and the motif has the sequence ofG-G-T-G-A-G-C-G (SEQ ID NO: 193). Examples of norovirus-binding aptamerscomprising motif 19 include AP5-GI (SEQ ID NO: 180) and AP6-GI (SEQ IDNO: 181).

Another family of norovirus-binding aptamers comprises motif 20. Motif20 is characterized by the sequence N₁-G-T-N₂-G-G (SEQ ID NO: 187),where N₁ and N₂ can be any nucleotide. Optionally, one or morenucleotides in the motif can be modified. In some cases, N₁ and N₂ caneach be A or T, and the motif has the sequence W₁-G-T-W₂-G-G (SEQ ID NO:194) using IUPAC nomenclature, where W can be A or T. In one example ofmotif 20, W₁ is A and W₂ is A, and the motif has the sequence ofA-G-T-A-G-G (SEQ ID NO: 195). In another example of motif 20, W₁ is Tand W₂ is T, and the motif has the sequence of T-G-T-T-G-G (SEQ ID NO:196). Examples of norovirus-binding aptamers comprising motif 20 includeAP3-GI (SEQ ID NO: 178) and AP6-GI (SEQ ID NO: 181).

Another family of norovirus-binding aptamers comprises motif 21. Motif21 is characterized by the sequence N₁-G-G-T-N₂-G (SEQ ID NO: 188),where N₁ and N₂ can be any nucleotide. Optionally, one or morenucleotides in the motif can be modified. In some cases, N₁ and N₂ caneach be A or C, and the motif has the sequence M₁-G-G-T-M₂-G (SEQ ID NO:197) using IUPAC nomenclature, where M can be A or C. In one example ofmotif 21, M₁ is A and M₂ is A, and the motif has the sequence ofA-G-G-T-A-G (SEQ ID NO: 198). In another example of motif 20, M₁ is Cand M₂ is C, and the motif has the sequence of C-G-G-T-C-G (SEQ ID NO:199). Examples of norovirus-binding aptamers comprising motif 21 includeAP2-GI (SEQ ID NO: 177) and AP6-GI (SEQ ID NO: 181).

Another family of norovirus-binding aptamers comprises motif 22. Motif22 is characterized by the sequence G-G-A-T-T-A-G (SEQ ID NO: 189).Optionally, one or more nucleotides in the motif can be modified.Examples of norovirus-binding aptamers comprising motif 22 includeAP1-GI (SEQ ID NO: 176) and AP5-GI (SEQ ID NO: 180).

Another family of norovirus-binding aptamers comprises motif 23. Motif23 is characterized by the sequence G-G-A-A-T-A-G (SEQ ID NO: 190).Optionally, one or more nucleotides in the motif can be modified.Examples of norovirus-binding aptamers comprising motif 23 includeAP1-GI (SEQ ID NO: 176).

Some norovirus-binding aptamers comprise one or more of motifs 1-14.Other norovirus-binding aptamers comprise norovirus-binding motifs otherthan motifs 1-14. Other norovirus-binding aptamers comprise one or moreof motifs 1-14 in combination with other norovirus-binding motifs. Somenorovirus-binding aptamers comprise one or more of motifs 1-23. Othernorovirus-binding aptamers comprise norovirus-binding motifs other thanmotifs 1-23. Other norovirus-binding aptamers comprise one or more ofmotifs 1-23 in combination with other norovirus-binding motifs.

B. Targets of Norovirus-Binding Aptamers

The norovirus-binding aptamers disclosed herein preferentially bind tonorovirus targets. In some cases, the norovirus-binding aptamers havelittle cross-reactivity with other viruses, such as hepatitis A virus(HAV) or enteroviruses such as poliovirus. The term “norovirus” includesintact noroviruses (aggregated or not aggregated) from all genogroups,genotypes, and strains of norovirus; norovirus virus-like particles; andparts or combinations of parts of intact noroviruses or norovirusvirus-like particles. Noroviruses are a group of small, geneticallydiverse viruses belonging to the genus Norovirus, family Caliciviridae.Noroviruses can be segregated genetically into at least five genogroups,I to V, based on the molecular characterization of the capsid genesequences (see Zheng et al., Virology 346:312-323 (2006)). Thegenogroups are further divided into more than 30 genotypes. Genotypescan further be divided into strains or variants. Strains of threegenogroups—GI, GII, and GIV—are found in humans, and the GIII and GVgenogroups are found in cows and mice, respectively. The GII genogroup,and more specifically variants of the GII.4 genotype, comprise theepidemic strains and cause the majority of outbreaks. However, otherstrains cause outbreaks as well and many of these are foodborne.Examples of norovirus strains include Snow Mountain virus (SMV;prototypical GII.2 norovirus), Norwalk virus (NV; prototypical GI.1norovirus), Houston virus (HOV; a GII.4 strain), and Grimbsy virus (GRV;a GII.4 strain).

An intact norovirus or norovirus particle is a norovirus that is notdisassembled in any significant way. In natural environments, norovirusparticles are frequently aggregated. See Teunis et al., J. Med. Viral.80:1468-1476 (2008). The term “virus-like particle” or “VLP” refers to astructure that in at least one attribute resembles a virus but which isnon-infectious and non-replicating. Virus-like particles are typicallymultiprotein structures that mimic the organization and conformation ofa native virus capsid, making them morphologically and antigenicallysimilar to a native virus, but are non-infectious because they lackviral genetic material sufficient to replicate. The expression of viralstructural proteins can result in the self-assembly of virus-likeparticles. A “part” of a norovirus or norovirus VLP can include, forexample, a portion of a norovirus (e.g., the viral capsid); a componentof the norovirus such as a particular norovirus protein; a domain orsubdomain within such a protein; an epitope within such a protein,domain, or subdomain; or an epitope spanning two or more proteins,domains, or subdomains. The term “target” or “target molecule” refers toany compound or molecule of interest to which binding of anorovirus-binding aptamer is desired.

Norovirus-binding aptamers can preferentially bind to various norovirustargets. In some cases, aptamers can preferentially bind to a particulargenogroup, genotype, or strain of norovirus or a target specific to aparticular genogroup, genotype, or strain of norovirus. For example, thetarget genogroup can be genogroup I, genogroup II, and/or othergenogroups, or the target genotype can be genogroup I, genotype 1;genogroup I, genotype 7; genogroup II, genotype 1; genogroup II,genotype 2; genogroup II, genotype 4; genogroup II, genotype 7; and/orother norovirus genotypes. Similarly, aptamers can preferentially bindto infectious norovirus particles or a target specific to infectiousnorovirus particles. As used herein, an “infectious” norovirus particleis a norovirus that has the capacity to invade an animal host andreplicate. In other cases, targets can be any combination of genogroups,genotypes, or strains of norovirus of or common to any combination ofgenogroups, genotypes, or strains of norovirus and/or common toinfectious and non-infectious norovirus particles.

Norovirus-binding aptamers typically bind to their designated norovirustarget with an affinity characterized by an equilibrium dissociationconstant (K_(d)) of less than about 10⁻⁴, less than about 10⁻⁵, lessthan about 10⁻⁶, less than about 10⁻⁷, less than about 10⁻⁸, less thanabout 10⁻⁹, less than about 10⁻¹⁰, less than about 10⁻¹¹, or less thanabout 10⁻¹² M. For example, some norovirus-binding aptamers can have anaffinity characterized by an equilibrium dissociation constant valuefrom about 0.001 nM to about 100 μM, from about 0.1 nM to about 999 nM,from about 1 nM to about 999 nM, from about 1 nM to about 500 nM, orfrom about 1 nM to about 300 nM.

Although smaller molecules such as proteins are typically used astargets for aptamers, complex heterogeneous targets such as whole cells,viruses, or virus-like particles are also employable for the generationof aptamers (see WO 2011/097420; Ohuchi, BioRes. Open Access 1:265-271(2012)). Thus, in some cases, the target can be a norovirus virus-likeparticle, an intact norovirus, an intact human norovirus, or aninfectious norovirus particle. This interaction can enable selection ofnorovirus-binding aptamers against these norovirus targets in theirnative conformation and physiological environment. Furthermore,norovirus-binding aptamers can be identified that preferentially bind tomultiple norovirus surface targets in their native conformations andphysiological environments, which can lead to candidatenorovirus-binding aptamers with different levels of specificity (e.g.,genogroups, genotypes, or strains) or even the ability to discriminatebetween different physiological states (e.g., infectious ornon-infectious).

Norovirus targets can also comprise norovirus proteins or any other partor combination of parts of an intact norovirus, intact human norovirus,infectious norovirus particle, or a norovirus virus-like particle. Majorand minor norovirus capsid proteins include VP1 and VP2, respectively(see Karst, Viruses 2:748-781 (2010); Hardy, FEMS Microbiol. Lett.253:1-8 (2005)). VP1 can range from about 530-555 amino acids, and VP2can range from about 208-268 amino acids. Norovirus capsids can beformed from 180 copies (90 dimers) of VP1 arranged with T=3 icosahedralsymmetry and one or two copies of VP2 (see Hardy, FEMS Microbiol. Lett.253:1-8 (2005); Taube et al., J. Virol. 84:5695-5705 (2010)). VP1 canself-assemble into virus-like particles (VLPs) in baculovirus,mammalian, and plant expression systems. The VP1 protein forms twodomains: P (protruding, P1 and P2) and S (shell). The norovirus genomealso encodes a nonstructural polyprotein, which can then be cleaved intoat least six different nonstructural proteins, including VPg, p48, p22,an NTPase, a protease, and an RNA-dependent RNA polymerase.

In some cases, the norovirus target to which a norovirus-binding aptamerpreferentially binds can be the viral capsid, the VPg protein or anyother nonstructural protein, the VP1 protein, the VP2 protein, anepitope within any of the above, an epitope spanning both capsidproteins, or a region of one of the VPg, VP1, or VP2 proteins necessaryfor a particular functionality. For example, the target can be the Sdomain, the P domain, the P1 subdomain, or the P2 subdomain within theVP1 protein, or an epitope within any of these domains or subdomains orspanning two or more of these domains or subdomains.

The term “epitope” refers to a site on a target to which an aptamerbinds. For example, an epitope on a protein target can be formed fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of one or more proteins. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents, whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast about 2, at least about 3, at least about 5, or at least about8-10 amino acids. Such amino acids can be in a specific spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, X-ray crystallography and two-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). Thus, anorovirus target can be, for example, an epitope comprising at leastabout 2, at least about 3, at least about 5, or at least about 8-10amino acids from the P2 subdomain of the P domain of the VP1 protein.

In some cases, only one norovirus target is selected, and thenorovirus-binding aptamer preferentially binds to that target. In othercases, more than one target molecule is selected. When more than onetarget is selected, the targets can be from the same genogroup,genotype, or strain of norovirus, or from different genogroups,genotypes, or strains of norovirus. In other cases, targets can alsoinclude norovirus targets and non-norovirus targets.

In some cases, norovirus targets to which norovirus-binding aptamerspreferentially bind are necessary for the function of the intactnorovirus such that aptamers binding to such targets will affect afunction of those molecules and/or a function of the intact norovirus.Such functions might include facilitating colonization of a niche in thehost (e.g., adhesion of norovirus to host cells such as enterocytes,macrophages, and dendritic cells), evasion of the host's immuneresponse, entry into or exit out of host cells, or production ofapoptotic or other toxic responses in host cells. For example,association of the norovirus with histo-blood group antigens (HBGAs),which are neutral carbohydrates linked to proteins or lipids on cellsurfaces, can be blocked. Many of the cellular interactions and immunerecognition features may be located in the norovirus VP1 protein, and inparticular the P2 subdomain, which is a subdomain that extends above theviral surface and has the most sequence divergence in the genome. Thus,the P2 subdomain can contain sites for antigenicity, immune-drivenevolution, and cell binding. In some cases, such sites can be used astargets selected to affect corresponding functions of the norovirus.

Another example of a norovirus target necessary for the function of theintact norovirus is the VPg protein. The VPg protein is covalently boundto HuNoV genomic and subgenomic RNA and can be involved in translationof viral RNA and in initiating transcription. The VPg protein has beenfound to be necessary for HuNoV replication (see Guix et al., J.Virology 81(22):12238-12248 (2007)). As such, it can be required forHuNoV to be infectious. Thus, the VPg protein can be selected as anorovirus target to affect these functions.

C. Modifications of Norovirus-Binding Aptamers

The norovirus-binding aptamers disclosed herein can contain modifiednucleic acids or other chemical or physical modifications. Suchmodifications can be made, for example, in order to increase the in vivostability of the aptamer, to enhance or mediate the delivery of theaptamer, or to reduce clearance rate of the aptamer from the body. Forexample, aptamers can be pretreated with an anti-nuclease before use.Such modifications can also include those which provide other chemicalgroups that incorporate additional charge, polarizability, hydrogenbonding, electrostatic interaction, and fluxionality to the nucleic acidbases or to the aptamer as a whole. Modifications can also be made tomake the aptamer as small as possible while still retainingnorovirus-binding activity, to increase specificity for the target, toconfer resistance to degradation, to provide capability to cross varioustissue or cell membrane barriers, or any other accessory properties thatdo not significantly interfere with affinity for the target (see, e.g.,U.S. Pat. No. 5,496,938).

Examples of such modifications include nucleotide substitutions, as wellas chemical substitutions at the deoxyribose/ribose and/or phosphateand/or base positions of a given nucleotide sequence (see, e.g., WO92/03568; U.S. Pat. Nos. 5,118,672; 5,660,985; 6,090,932). Aptamers caninclude one or more substitute internucleotide linkages, altered sugars,altered bases, or combinations thereof (see, e.g., WO 2011/061351). Forexample, such aptamers can contain one or more nucleotides with2′-position sugar modifications such as 2′-amino (2′-NH₂), 2′-O-methyl(2′-OMe), and/or 2′-fluoro (2′-F) groups. Such aptamers can also containnucleotide derivatives chemically modified at the 5′ and 2′ positions ofpyrimidines or at the 8′ position of purines (see U.S. Pat. Nos.5,660,985 and 5,580,737). Other modifications include modifications atexocyclic amines, substitution of 5-bromo-uracil or 5-iodo-uracil,substitution of 4-thiouridine, backbone modifications, methylations,unusual base-pairing combinations such as the isobases isocytidine andisoguanidine, and the like. Modifications can also include 3′ and 5′modifications such as capping. Some specific modifications include the5′-5′ inversion, 5′-cholesteryl, 5′-liposome, 3′-3′ inversion, 3′-Tcapping, 3′-biotin, mirror-design, phosphorothioate replacement,mehtylphosphonate replacement, N3′-P5′, locked nucleic acid (LNA),unlocked nucleic acid (UNA), hexitol nucleic acid (HNA),2′-O-methyl-4′-thio, 5-N-(6-aminohexyl) carbamoyl-2′-deoxyuridine,4′-C-(aminoethyl) thymidine, and 4′-thio modifications described in Wanget al., Curr. Med. Chem. 18:4126-4138 (2011).

The norovirus-binding aptamers disclosed herein can also be coupled toother elements. Such elements can, for example, add independent affinityfor the target, enhance the affinity of the aptamer to the target,direct or localize the aptamer to the desired location in vivo, orutilize the specificity of the aptamer to the target to effect someadditional reaction at that location.

Some norovirus-binding aptamers are chimeric aptamers. Such aptamers,and methods for making them, are described in U.S. Pat. No. 5,637,459.Chimeric aptamers are aptamers with two or more functionalities.Chimeric norovirus-binding aptamers can comprise two or more componentaptamers. In some cases, the component aptamers are 3′-3′-linked. Somechimeric norovirus-binding aptamers preferentially bind to differentepitopes of the same norovirus target. For example, a chimericnorovirus-binding aptamer can comprise one component norovirus-bindingaptamer targeting a first epitope within the P1 domain of a norovirusand a second component norovirus-binding aptamer targeting a secondepitope within the P1 domain of a norovirus. Other chimericnorovirus-binding aptamers preferentially bind to epitopes on twodifferent targets on the same norovirus. For example, a chimericnorovirus-binding aptamer can comprise one component norovirus-bindingaptamer targeting the VP1 protein of a norovirus and a second componentnorovirus-binding aptamer targeting the VP2 protein of a norovirus.Other chimeric norovirus-binding aptamers preferentially bind to anepitope on a target norovirus and an epitope on a non-norovirusmolecule.

Some norovirus-binding aptamers are blended aptamers. Such aptamers, andmethods for making them, are described in U.S. Pat. No. 5,683,867.Blended norovirus-binding aptamers comprise a norovirus-binding aptamercoupled to at least one other functional unit. The functional unit canbe coupled to the 5′ end of the aptamer, the 3′ end of the aptamer, orany other location within the norovirus-binding aptamer that does notaffect a desired functionality of the norovirus-binding aptamer. Someexamples of functional units include peptides, amino acids, aliphaticgroups and lipid chains, or peptide motifs. In some cases, thefunctional units are recognizable by the norovirus target. For example,the functional unit may fit into a specific binding pocket on thenorovirus target to form tight binding via hydrogen bonds, salt bridges,or van der Waals interactions. Some functional units guide thenorovirus-binding aptamers to specific sites on the target.

Other functional units add functionality to the norovirus-bindingaptamer, for example, to increase RNA hydrophobicity and enhancebinding, membrane partitioning, and/or permeability, or to add reportermolecules or labels, such as biotin- or fluorescence-tagged reporteroligonucleotides. Examples of such labels include radiolabels,fluorophores, chromophores, and affinity tags. Such labels can beradiolabels used for medical imaging (e.g., technetium-99m (tc99m) oriodine-123 (1123)), or spin labels used for nuclear magnetic resonance(NMR) imaging or magnetic resonance imaging (MRI), such as iodine-123,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese, or iron. Examples of fluorophores includefluorescein, rhodamine, Cy5 reactive dye, Cy3 reactive dye,allophycocyanin, peridinine chlorophyll-a protein (PerCP),phycoerythrin, Alexa Fluor, and green fluorescent protein (GFP). Otherlabels include quantum dots (QDs), which are nanocrystals made ofsemiconductor materials that function like fluorophores. Functionalgroups can also comprise chemical groups which covalently react andcouple the norovirus-binding aptamer to the norovirus target.

Several approaches can be used to create chimeric or blendednorovirus-binding aptamers. Such approaches include conjugationapproaches involving 3′ or 5′ primary amines coupled to various othermolecules during chemical synthesis of nucleic acids, or classiccarbodiimide, aldehyde, diazonium, or other approaches which takeadvantage of the greater chemical reactivity or primary alkyl amine tagsversus aryl amines on the nucleotides themselves. Such approaches, aswell as several types of aptamer-drug and aptamer-toxin conjugates, aredescribed in Bruno, Pharmaceuticals 6:340-357 (2013).

The norovirus-binding aptamers disclosed herein can be coupled to otherelements through direct covalent conjugation. Direct conjugation can beachieved, for example, by construction of a polynucleotide fusionthrough genetic fusion of two component aptamers so that they exist as asingle polynucleotide. Direct conjugation can also be achieved byformation of a covalent bond between a reactive group on anorovirus-binding aptamer and a corresponding group or acceptor on acoupled element. Direct conjugation can also be achieved by modificationof one of the two molecules to be conjugated to include a reactive groupthat forms a covalent attachment to the other molecule to be conjugatedunder appropriate conditions. For example, methods for covalentconjugation of nucleic acids to proteins are known (e.g.,photocrosslinking, see, e.g., Zatsepin et al., Russ. Chem. Rev. 74:77-95(2005)).

Coupling may also be performed using a variety of linkers. Linkers canbe any suitable spacer moiety. For example, suitable linker groupsinclude polynucleotides, PEG, polyvinyl alcohol, polyacrylates, andpolypeptides. In some cases, the linkage is cleavable. Examples ofsuitable cleavable linkages include photochemically labile linkers,disulfides, and carbonates. The linkage can also be cleavable withenzymes, such as DNAses and proteinases. Linkers that release the linkedelement under acidic or reducing conditions can also be employed.

The norovirus-binding aptamers disclosed herein can also be tethered toa solid support. For example, the aptamers can be immobilized onmagnetic beads. This can be achieved, for example, onP(HPMA/EGDMA)-g-p(GMA) magnetic beads via glutaraldehyde linking (seeOzalp et al., Anal. Biochem. 447:119-125 (2014) or using biotinylatedaptamers and streptavidin paramagnetic particles. Norovirus-bindingaptamers can also be immobilized on gold electrode chips (see Kim etal., Biosensors and Bioelectronics 54:195-198 (2014)) or on any othersolid support that is suitable for a desired application, such as amethod of detection, capture, or concentration of norovirus.

D. Combinations Comprising Norovirus-Binding Aptamers

Norovirus-binding aptamers disclosed herein can also be combined withbut not physically coupled to other aptamer(s) and/or element(s) in onemixture. Optionally, the norovirus-binding aptamer and other aptamer(s)and/or element(s) can be differentially labeled. The term“differentially labeled” indicates that the norovirus-binding aptamerand other aptamer(s) and/or molecule(s) can be distinguished from eachother because each is coupled to a different label.

In some cases, a mixture of norovirus-binding aptamers may be generatedin which the different component aptamers preferentially bind to thesame norovirus target(s). The component norovirus-binding aptamers may,for example, preferentially bind to different epitopes on the samenorovirus target. In some cases, the ability of a mixture ofnorovirus-binding aptamers to bind to different epitopes on the samenorovirus target will result in increased binding affinity to thenorovirus target compared to norovirus-binding aptamers binding to onlyone epitope.

In other cases, a mixture of norovirus-binding aptamers may be generatedin which the different component aptamers preferentially bind todifferent norovirus targets. For example, such mixtures can include oneor more norovirus-binding aptamers that preferentially bind to one ormore genogroups of noroviruses and one or more other norovirus-bindingaptamers that preferentially bind to one or more different genogroups ofnoroviruses. Similarly, such mixtures can include one or morenorovirus-binding aptamers that preferentially bind to one or moregenotypes of noroviruses and one or more other norovirus-bindingaptamers that preferentially bind to one or more different genotypes ofnoroviruses. Likewise, such mixtures can include one or morenorovirus-binding aptamers that preferentially bind to one or morestrains of norovirus and one or more other norovirus-binding aptamersthat preferentially bind to one or more different strains of norovirus.

In yet other cases, a mixture of aptamers may be generated in which oneor more component aptamers are norovirus-binding aptamers thatpreferentially bind to a norovirus target and one or more other aptamerspreferentially bind to a non-norovirus target. For example, suchmixtures can include one or more aptamers that preferentially bind tonoroviruses and one or more other aptamers that preferentially bind toanother type of virus or bacteria. In some cases, the other type ofvirus or bacteria is a pathogenic virus or bacteria, and the one or moreaptamers that preferentially bind to noroviruses and the one or moreother aptamers that preferentially bind to another type of virus orbacteria are differentially labeled such that the noroviruses and theother type of virus or bacteria can be differentially detected.

The norovirus-binding aptamers disclosed herein can also be combinedwith but not physically coupled to one or more other non-aptamermolecules in a mixture. In some cases, the other non-aptamer moleculespreferentially bind to norovirus targets. In other cases, the othernon-aptamer molecules preferentially bind to non-norovirus targets. Insome cases, the other non-aptamer molecules can be norovirus-bindingmolecules, such as antibodies or peptide aptamers. Such othernon-aptamer molecules can bind to the same target as thenorovirus-binding aptamer. For example, the norovirus-binding aptamercan bind to one epitope on the norovirus target, and the othernon-aptamer molecule can bind to another epitope on the same norovirustarget. Such other non-aptamer molecules can also bind to a differentnorovirus target or a non-norovirus target. For example, such mixturescan include norovirus-binding aptamers that preferentially bind to oneor more genogroups of noroviruses and other non-aptamer molecules thatpreferentially bind to one or more different genogroups of noroviruses.Similarly, such mixtures can include norovirus-binding aptamers thatpreferentially bind to one or more genotypes of noroviruses and othernon-aptamer molecules that preferentially bind to one or more differentgenotypes of noroviruses. Likewise, such mixtures can includenorovirus-binding aptamers that preferentially bind to one or morestrains of norovirus and other non-aptamer molecules that preferentiallybind to one or more different strains of norovirus.

III. Methods for Detecting Presence of, Capturing, and/or ConcentratingNorovirus

The norovirus-binding aptamers disclosed herein can be used forspecifically, qualitatively, and/or quantitatively detecting norovirusfrom any source in the context of clinical diagnosis, treatment, and/orresearch based on preferential binding of the aptamer to the norovirus.For example, the aptamers can be used for detecting norovirus in a testsample as an indication that the test sample contains norovirus.

Different types of test samples can be used. The term “test sample”refers to any clinical sample, environmental sample, food sample, or anyother type of sample that is being assayed for the presence ofnorovirus. A test sample can be taken or derived from anything oranywhere susceptible to contain norovirus. Some test samples can be useddirectly. Other test samples can be subjected to purifying protocols orother processing, such as coating on plates.

Some test samples can comprise captured or pre-concentrated norovirus.The term “captured norovirus” refers to norovirus that has beensubstantially separated from a test sample. For example, a test samplecan be contacted with a norovirus-binding aptamer disclosed herein orany other norovirus-binding molecule, and the bound norovirus can thenbe substantially separated from the remainder of the test sample. Thenorovirus that has been substantially separated from the remainder ofthe test sample is the captured norovirus. Both the captured norovirusand the original test sample from which the captured norovirus wassubstantially separated are considered to be test samples.

As used herein, the term “substantially separating” refers to removing amolecule of interest (e.g., a norovirus) so that it is at leastpartially free of one or more other components with which it waspreviously associated. For example, a norovirus of interest can be atleast partially free of some non-norovirus components with which it waspreviously associated but not other non-norovirus components. In somecases, the molecule of interest is at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, or 100% free of one or more othercomponents with which it was previously associated. For example,substantially separating aptamer-bound norovirus from a test samplemeans that the aptamer-bound norovirus (aggregated or not aggregated) isat least about 30%, at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,or 100% free of one or more other components present in the test sample.

The term “clinical sample” refers to a sample of biological materialwithin or obtainable from a patient or any other biological source, suchas another human or mammalian subject. Such samples can include, forexample, organs, organelles, tissues, sections of tissues, bodilyfluids, peripheral blood, blood plasma, blood serum, fecal matter,vomitus, urine, sputum, saliva, bronchial aspirate, cells, moleculessuch as proteins and peptides, and any parts or combinations derivedtherefrom. The term clinical sample can also encompass any materialderived by processing the sample. Derived material can include cells ortheir progeny. Processing of the clinical sample may involve one or moreof filtration, distillation, extraction, concentration, fixation,inactivation of interfering components, and the like. In certain cases,clinical samples are blood, fecal matter, vomitus, or any bodily fluidsample taken from a patient suffering or suspected to be suffering fromnorovirus infection.

The term “environmental sample” refers to a sample taken or derived fromany particular environment. For example, an environmental sample can betaken or derived from water (e.g., freshwater, salt water, waste water,and drinking water), soil, sewage, sludge, or an organism or tissue froman organism, such as a shellfish, that is a potential reservoir for anorovirus. An environmental sample can also be a sample taken or derivedfrom a particular surface or object (“fomite”) in a specific location,such as a restaurant, cruise ship, hospital, nursing home, school, orother location. In certain cases, an environmental sample is taken orderived from a particular environment suspected to be a source ofnorovirus.

The term “food sample” refers to a sample taken or derived from any foodor beverage product. Examples of such food or beverage products includefresh produce, deli meats, salad bars, prepared foods (e.g., sandwiches,meat salads, casseroles), raw and cooked molluscan shellfish, and highlyacidic foods such as orange juice and frozen raspberries. In certaincases, a food sample is taken from a food or beverage product suspectedto be a source of norovirus.

Some methods of detecting norovirus comprise contacting a test samplewith a norovirus-binding aptamer disclosed herein, and detecting thepresence of the norovirus-binding aptamer bound to norovirus in the testsample. Detection of bound aptamer can indicate the presence of at leastone norovirus strain. In some such methods, unbound aptamer is removedbefore detecting the presence of the norovirus-binding aptamer bound tonorovirus in the test sample. In some such methods, thenorovirus-binding aptamers disclosed herein can be labeled withfluorescent molecules, spin-labeled molecules, enzymes, radioisotopes,or similar labels, and they can also be provided in the form of a kitwith all the necessary reagents to perform the assay for detection ofnorovirus.

In some methods of detecting norovirus, a test sample is contacted witha norovirus-binding aptamer disclosed herein under conditions and for anamount of time sufficient to permit the norovirus-binding aptamer tobind to the norovirus. For example, a test sample can be incubated withat least about 10 nM, at least about 100 nM, at least about 500 nM, atleast about 1 or at least about 2 μM of norovirus-binding aptamer. Insome cases, the incubation can be done at room temperature. In othercases, it can be done at 4° C. In some cases, the incubation can beovernight. In other cases, the incubation can be for at least about 5minutes, at least about 10 minutes, at least about 20 minutes, at leastabout 30 minutes, at least about 45 minutes, at least about 1 hour, atleast about 2 hours, at least about 3 hours, at least about 4 hours, atleast about 5 hours, at least about 6 hours, at least about 12 hours, orat least about 24 hours.

In some methods of detecting norovirus, unbound aptamer is removed aftercontacting the test sample with the norovirus-binding aptamer. Unboundaptamer may be removed (e.g., by washing one or more times with, forexample, a buffer) under conditions such that some of thenorovirus-binding aptamer that is bound to norovirus will remain boundto the norovirus. For example, unbound aptamer can be removed by washingone or more times with a buffer. In some cases, substantially all of thenorovirus-binding aptamer that is bound to norovirus will remain boundto the norovirus.

Many assays can be used to qualitatively or quantitatively detectbinding of the norovirus-binding aptamers disclosed herein to norovirus.For example, an Enzyme-Linked Aptamer Sorbent Assay (ELASA) can be used.Assays involving amplification of the bound aptamer (e.g. qPCR) or RNAfrom the aptamer-bound virus (e.g., qRT-PCR) can be used. Flow cytometrymethods as described in U.S. Pat. No. 5,853,984 can be used.Microarrays, BIAcore assays, differential centrifugation,chromatography, electrophoresis, immunoprecipitation, opticalbiosensors, and other surface plasmon resonance assays can be used asdescribed in WO 2011/061351. Other assays that can be used arecalorimetric analysis and dot blot assays. Moreover, just as theenzyme-linked immunosorbent assay (ELISA) was adapted for aptamers inthe ELASA assay, any other assays involving norovirus-binding antibodiescan be adapted for use with the norovirus-binding aptamers disclosedherein in place of the antibodies. Such assays include immunometricassays such as radioimmunoassays, flow cytometry assays, blottingapplications, anisotropy, membrane assays, biosensors, and the like. Anyother assays known in the art can also be used or adapted.

Methods of detecting norovirus using the norovirus-binding aptamersdisclosed herein can also be combined with other methods of detectingnorovirus. For example, methods of detecting norovirus using thenorovirus-binding aptamers disclosed herein can be combined with sampleconcentration and amplification of viral RNA as described in U.S. Pat.No. 7,205,112, or any methods utilizing norovirus-binding antibodies,including immunometric assays such as enzyme-linked immunosorbent assays(ELISAs), radioimmunoassays, flow cytometry assays, blottingapplications, anisotropy, membrane assays, biosensors, and the like.

Methods for detecting norovirus can have varying degrees of inclusivityand exclusivity. In some cases, the norovirus-binding aptamers used areexclusive, wherein only noroviruses of one or more particulargenogroups, genotypes, or strains can be detected. In other cases, thenorovirus-binding aptamers used are more inclusive, wherein norovirusesof a wide range of genogroups, genotypes, or strains of norovirus can bedetected. If multiple genogroups, genotypes, or strains can be detected,particular genogroups, genotypes, or strains may be preferentiallydetected due to a higher binding affinity of the norovirus-bindingaptamer(s) to those particular genogroups, genotypes, or strains.Methods of detecting norovirus can also be specific for infectiousnorovirus particles or alternatively can detect both infectious andnon-infectious norovirus particles.

In some cases, only infectious norovirus particles can be detected. Forexample, norovirus-binding aptamers can be used that preferentially bindto intact norovirus capsids, VPg, or another protein whose function isrequired for norovirus to be infectious.

Some methods of detecting norovirus can differentially detect two ormore genogroups, genotypes, or strains of norovirus or candifferentially detect infectious and non-infectious norovirus. Forexample, a mixture of norovirus-binding aptamers can be used, whereinthe first norovirus-binding aptamer preferentially binds to one or moregenogroups, genotypes, or strains of norovirus and one or more othernorovirus-binding aptamers preferentially bind to one or more differentgenogroups, genotypes, or strains of norovirus. The differentnorovirus-binding aptamers can be differentially labeled, and detectionof bound first aptamer and bound other aptamers can indicate thepresence of at least two norovirus genogroups, genotypes, or strains ina test sample. Comparable methods of detecting two or more genogroups,genotypes, or strains of norovirus involve use of a norovirus-bindingaptamer disclosed herein that preferentially binds to one or moregenogroups, genotypes, or strains of norovirus, and one or more otherdetection compounds (e.g., peptide aptamers, antibodies, or the like)that detect one or more different genogroups, genotypes, or strains ofnorovirus.

Some methods of detecting norovirus can further detect non-norovirustargets as well. For example, a norovirus-binding aptamer disclosedherein can be used with one or more other detection compounds (e.g.,nucleic acid aptamers, peptide aptamers, antibodies, or the like) thatpreferentially bind to other types of viruses or bacteria, such ashepatitis A virus or Listeria monocytogenes (see, e.g., U.S. Pat. No.7,645,582). In some cases, the other types of viruses or bacteriadetected with the one or more other detection compounds can causesymptoms in a patient that are similar to those caused by norovirusinfection. The norovirus-binding aptamer and the other detectioncompound(s) can be differentially labeled so that detection of boundaptamer can indicate norovirus infection whereas detection of binding ofthe other detection compound(s) can indicate a different type ofinfection or condition.

Binding of the aptamers in a test sample can be compared to binding ofthe aptamers in a control sample. The term “control sample” refers to atest sample, clinical sample, environmental sample, or food sample notknown or suspected to include norovirus, or not known or suspected toinclude norovirus of a given genogroup, genotype, or strain. Suchsamples can be obtained at the same time as a test sample, clinicalsample, environmental sample, or food sample suspected to includenorovirus, or they can be obtained on a different occasion. Such samplescan be obtained from the same source or from different sources. Thecontrol sample can be the same type of sample as the test sample,clinical sample, environmental sample, or food sample to which it isbeing compared. For example, if the clinical sample comprises fecalmatter, then the control sample can comprise fecal matter. Similarly, ifthe food sample comprises lettuce, then the control sample can compriselettuce.

Multiple test samples and multiple control samples can be evaluated onmultiple occasions to protect against random variation independent ofthe differences between the samples. A direct comparison can then bemade between the test samples and the control samples to determinewhether aptamer binding (i.e., the presence of norovirus) in the testsamples is increased, decreased, or the same relative to aptamer bindingin the control samples. Increased binding of the aptamer in the testsamples relative to the control samples indicates the presence of atleast one strain of norovirus in the test samples. In some instances,increased binding is statistically significant relative to the controlsample. For example, statistical significance can mean p≤0.1, p≤0.05,p≤0.01, or p≤0.001. In some cases, the term “increased binding” refersto aptamer binding in a test sample that is at least about 1.5-fold, atleast about 2-fold, at least about 3-fold, at least about 4-fold, atleast about 5-fold, at least about 10-fold, or at least about 20-foldhigher than aptamer binding in a control sample.

The norovirus-binding aptamers disclosed herein can also be used forcapturing, purifying, and/or concentrating norovirus from test samples.Such methods can comprise contacting a test sample with anorovirus-binding aptamer disclosed herein and then substantiallyseparating the aptamer-bound norovirus from the remainder of the testsample. For example, norovirus can be captured with nanomagneticstreptavidin beads using biotinylated aptamers.

The norovirus that was substantially separated from the remainder of thetest sample (i.e., the captured norovirus) can be in a more concentratedform than the norovirus in the original test sample. For example, theconcentration of the captured norovirus can be at least about 1.5-fold,at least about 2-fold, at least about 3-fold, at least about 4-fold, atleast about 5-fold, at least about 10-fold, at least about 20-fold, atleast about 25-fold, at least about 30-fold, at least about 40-fold, atleast about 50-fold, at least about 75-fold, or at least about 100-foldhigher than the norovirus concentration in the original test sample.Norovirus concentration can be calculated, for example, in terms ofgenomic copies of viral RNA per liter or per gram of sample (e.g., usingqRT-PCR).

Captured and/or concentrated norovirus can be used to facilitatedetection using molecular amplification methods. For example, an aptamerbound to the captured norovirus can be used for subsequent qPCRamplification as an indirect method of detecting the norovirus. Othermolecular amplification methods that can be used include viral RNAextraction and subsequent real-time RT-PCR. Other nucleic acidamplification methods such as RT-LAMP (Reverse Transcription-LoopMediated Isothermal Amplification), RPA (Recombinase PolymeraseAmplification), and others could also be used.

Methods of capturing and/or concentrating norovirus using thenorovirus-binding aptamers disclosed herein can be combined with othernorovirus concentration and purification schemes. Such other schemes canbe based on sample manipulations that capitalize on the behavior ofenteric viruses to be released from particulates at high pH and saltconcentration (elution), to act as proteins in solutions (precipitationusing polyethylene glycol), to co-sediment by simple centrifugation whenadsorbed to larger particles, and to remain infectious at pH extremes orin the presence of organic solvents. Filtration and ultracentrifugationcan also be used. Other schemes that can be used involve capture byantibodies or carbohydrate ligands. Examples of ligands that can be usedinclude porcine gastric mucin, histo-blood group antigens (HBGAs),HBGA-like substances, and human plasma protein components.

Similarly, methods of capturing and/or concentrating norovirus using thenorovirus-binding aptamers disclosed herein can be combined withnorovirus detection schemes such as antibody-based detection, or methodsof detecting norovirus using the norovirus-binding aptamers disclosedherein can be combined with other virus capturing and/or concentratingschemes. One example of a method of detecting norovirus using thenorovirus-binding aptamers disclosed herein combined with another viruscapturing scheme is the two-site binding sandwich qPCR assay. In suchassays, any aptamer-based or non-aptamer-based methods of capturingnorovirus can be used prior to exposing a norovirus-binding aptamerdisclosed herein to the captured norovirus. In some such assays, themolecule(s) used for the initial capture of the norovirus can bind to anepitope on the norovirus that is different from the epitope to which thenorovirus-binding aptamer binds. After exposing the captured norovirusto the norovirus-binding aptamer, the bound norovirus-binding aptamercan be detected. For example, the bound norovirus-binding aptamer can beamplified by qPCR.

IV. Methods for Identification and Selection of Aptamers

Methods are provided herein for the identification and selection ofaptamers that preferentially bind to norovirus targets. Some suchmethods comprise the Systematic Evolution of Ligands by EXponentialEnrichment (SELEX) method or modifications thereof. SELEX is a methodfor selecting high affinity ligands of a target. The SELEX method isdescribed in detail elsewhere. See, e.g., U.S. Pat. Nos. 5,270,163;5,567,588; 5,696,249; and 5,853,984, herein incorporated by reference intheir entirety for all purposes.

A SELEX process for identifying norovirus-binding aptamers can comprisecontacting a candidate mixture of nucleic acids with a selectednorovirus target and partitioning the nucleic acids with relativelyhigher affinity to the target from the nucleic acids with relativelylower affinity to the target. In some cases, the nucleic acids thatpreferentially bind to the selected target can then be amplified. Anycombination of the steps can be repeated as desired until the desirednumber and affinity of norovirus-binding aptamers is achieved. Byrepeating the partitioning and amplifying steps, the newly formedcandidate mixture may contain fewer and fewer unique sequences, and theaverage degree of affinity of the nucleic acids to the norovirus targetcan increase. The SELEX process can yield a candidate mixture containingone or a small number of unique nucleic acid sequences representingthose nucleic acids from the original candidate mixture, or portionsthereof, having the highest affinity to the norovirus target.

The term “candidate mixture” refers to a mixture of nucleic acids ofdiffering sequence from which to select a desired norovirus-bindingaptamer that binds to a norovirus target with greater affinity than thatof the bulk population of nucleic acids or a desired norovirus-bindingaptamer that binds to a norovirus target with greater affinity than theaverage binding affinity of all of the nucleic acids in the candidatemixture. The source of a candidate mixture can be from naturallyoccurring nucleic acids or fragments thereof, chemically synthesizednucleic acids, enzymically synthesized nucleic acids, or nucleic acidsmade by a combination of these techniques. The candidate mixture maycontain nucleic acids with one or more types of modified nucleotides.Alternatively, modified nucleotides may be incorporated intonorovirus-binding aptamers that were identified by SELEX using acandidate mixture of nucleic acids not containing modified nucleotides.

The term “partitioning” refers to any process whereby norovirus-bindingaptamers bound to norovirus targets (hereinafter referred to asaptamer-target pairs) can be separated from nucleic acids not bound tonorovirus targets. Because only a small number of sequences (andpossibly only one molecule of nucleic acid) corresponding to the highestaffinity nucleic acids may exist in the candidate mixture, it can bedesirable to select partitioning criteria so that an appropriate amountof the nucleic acids in the candidate mixture (about 5-50%) can beretained during partitioning, providing a greater number ofnorovirus-binding aptamers. Partitioning can be accomplished by variousmethods known in the art. For example, nucleic acid-protein pairs can bebound to nitrocellulose filters while unbound nucleic acids are not.Columns that specifically retain aptamer-target pairs (or specificallyretain bound aptamer complexed to an attached norovirus target) can beused for partitioning. Liquid-liquid partition can also be used as wellas filtration gel retardation and density gradient centrifugation. Thechoice of partitioning method will depend on properties of the norovirustarget and of the aptamer-target pairs and can be made according toprinciples and properties known to those of ordinary skill in the art.

The term “amplifying” refers to any process or combination of steps thatincreases the amount or number of copies of a molecule or class ofmolecules. Any reaction or combination of reactions known in the art canbe used for amplifying as appropriate, including direct DNA replication,direct RNA amplification, and the like. The amplification method shouldresult in the proportions of the amplified mixture being representativeof the proportions of different sequences in the initial mixture.

Some methods for identification and selection of norovirus-bindingaptamers comprise the counter-SELEX method or modifications thereof. SeeU.S. Pat. Nos. 5,580,737 and 6,376,190, hereby incorporated by referencein their entirety for all purposes. Counter-SELEX can increasespecificity of SELEX pools and can decrease the number of SELEXselection rounds required to identify a norovirus-binding aptamerspecific for a norovirus target. Further, it provides a methodology foridentifying a norovirus-binding aptamer that does not cross-react withother molecules, including closely related molecules.

A method of using the SELEX and counter-SELEX processes to identifynorovirus-binding aptamers can be done in sequential steps. In SELEX, acandidate mixture of nucleic acids is placed in contact with a selectednorovirus target, partitioning the nucleic acids with relatively higheraffinity to the norovirus target from the nucleic acids with relativelylower affinity to the norovirus target, and amplifying the former. Incounter-SELEX, a nucleic acid pool is placed in contact with a samplecontaining one or more non-target molecules, and the nucleic acidsbinding to these non-targets are removed. The resulting non-boundnucleic acid mixture is then preferentially amplified. Any combinationof SELEX and counter-SELEX steps can be repeated as desired until thedesired number and affinity of norovirus-binding aptamers is achieved.

Any combination of rounds of SELEX and rounds of counter-SELEX can beused in any order. Thus, in some cases, SELEX and counter-SELEX roundsare not done in sequence. For example, one or more rounds of SELEX canbe performed, followed by one or more rounds of counter-SELEX, or viceversa.

Examples of non-target molecules that can be used to enhancenorovirus-binding aptamer specificity are a matrix used to immobilizethe virus during the SELEX process (e.g., antibody-bound beads),glutathione sepharose 4B, clinical samples such as human stool specimensthat are negative for norovirus, food samples negative for norovirus,environmental samples negative for norovirus, bacteria derived fromclinical samples (e.g., fecal matter), bacteria derived from foodsamples, bacteria derived from environmental samples, or unrelatedviruses such as hepatitis A virus or an enterovirus such as poliovirus.

There are many potential applications of the SELEX and counter-SELEXprocesses. For example, the selected norovirus target can correspond toproteins or protein structures that must be intact or complete in orderfor a norovirus particle to be infectious and the non-target moleculescan be those lacking the necessary structure(s), thereby producingnorovirus-binding aptamers that preferentially bind to infectiousnorovirus particles. Non-infectious viral particles can potentially begenerated, for example, through thermal inactivation of norovirus,chemical inactivation (e.g., 2% trisodium phosphate, 2% glutaraldehyde,1,000-5,000 ppm chlorine, etc.), electron beam inactivation, UVirradiation, gamma irradiation, high pressure processing, use of ozone,and pH inactivation (see Richards, Food Environ. Virol. 4:6-13 (2012)).In certain cases, thermal inactivation can be achieved by treatment at atemperature of at least 60° C., at least 65° C., at least 70° C., atleast 75° C., or at least 80° C. In some cases, thermal inactivation canbe achieved at a temperature of 63° C. or at a temperature of 65° C. Insome cases, the thermal inactivation treatment occurs over a period ofat least 0.5 minutes, at least 1 minute, at least 2 minutes, at least 3minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, atleast 7 minutes, at least 8 minutes, at least 9 minutes, at least 10minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes,or at least 30 minutes.

In other cases, the selected target can be norovirus strains from atarget genogroup and/or genotype, and the non-target molecules can benorovirus strains from other genogroups and/or genotypes, therebyproducing norovirus-binding aptamers that preferentially bind to thetarget genogroup and/or genotype. For example, the target genogroup canbe genogroup I, genogroup II, and/or other genogroups, or the targetgenotype can be genogroup I, genotype 1; genogroup I, genotype 7;genogroup II, genotype 1; genogroup II, genotype 2; genogroup II,genotype 4; genogroup II, genotype 7; and/or other norovirus genotypes.The selected target can also be a particular norovirus strain, with thenon-target molecules being any other norovirus strain, thereby producingnorovirus-binding aptamers that preferentially bind to the targetnorovirus strain. In other cases, the target can be a particular part ofa norovirus, thereby producing norovirus-binding aptamers thatpreferentially bind to the target part of the norovirus. For example,the target could be the VPg protein or any other nonstructural protein,the VP1 protein, the P domain within the VP1 protein, the P1 subdomainwithin the VP1 protein, the P2 subdomain within the VP1 protein, the Sdomain within the VP1 protein, or the VP2 protein. The target can alsobe a defined epitope within any of the above proteins, domains, orsubdomains, thereby producing norovirus-binding aptamers thatpreferentially bind to that epitope. The non-target molecules could thenbe, for example, any protein, domain, subdomain, or epitope other thanthe target protein, domain, subdomain, epitope.

A variation of the SELEX process is a method that uses graphene or aderivative thereof, such as graphene oxide. Because graphene and itsderivative graphene oxide have useful properties such as the ability toadsorb ssDNA and to release that DNA in the presence of specific targetsof the DNA, graphene and its derivatives, such as graphene oxide, can beused with SELEX to provide an immobilization-free platform for screeningof aptamers that bind to their target with high affinity and specifcity.For example, a pool of aptamers can be incubated with graphene oxide sothat they adsorb. The unadsorbed aptamers are then discarded, and theadsorbed aptamers can be incubated with a selected norovirus target forspecific desorption of the aptamers.

Secondary selection methods can be combined with SELEX and/orcounter-SELEX to select for additional functionality, such as ability tomodify a norovirus target function upon binding to selectednorovirus-binding aptamers. For example, such methods can includeselections or screens for inhibition, alteration of substrate binding,loss of functionality, disruption of structure, and the like. Those ofordinary skill in the art are able to select from among variousalternatives those selection or screening methods that are compatiblewith the methods described herein.

V. Methods for Assessing Efficacy of Therapeutic Agents or Vaccines

The instant disclosure further provides methods of evaluating theefficacy of a therapeutic agent in a patient diagnosed with a norovirusinfection. A “patient” includes a human or other mammalian subject thatreceives either prophylactic or therapeutic treatment. Treatment isconsidered prophylactic if administered to an individual susceptible toor otherwise at risk of norovirus infection. Treatment is consideredtherapeutic if administered to an individual suspected of having, oralready suffering from, norovirus infection.

An individual is at increased risk of norovirus infection if theindividual has at least one known risk factor placing individuals withthat risk factor at a statistically significant greater risk ofdeveloping an infection than individuals without the risk factor.Genetic risk factors such as histo-blood group antigen type or fucosyltransferase secretor status can mediate inherent susceptibility. Otherrisk factors can include, for example, presence on cruise ships or atother vacation settings, restaurants, hospitals, nursing homes, schools,or other locations where others have experienced norovirus infection orwhere a norovirus outbreak is suspected. Infected individuals can remaincontagious up to 48-72 hours after symptoms subside and perhaps longer.Some infections are asymptomatic, but infected individuals who areasymptomatic may still spread the virus. As few as 10 to 100 virusparticles are sufficient to cause infection, and the virus can surviveunder varying temperatures for days to months. Transmission is primarilythrough the fecal-oral route, direct person-to-person contact, byexposure to ill individuals experiencing vomiting, or by touchingcontaminated surface, objects, or substances. Risk factors can alsoinclude being exposed to clinical, food, or environmental samplespotentially contaminated with norovirus. Food-related norovirusoutbreaks have been associated with multiple products, such as freshproduce, deli meats, salad bars, prepared foods, raw and cookedmolluscan shellfish, highly acidic foods such as orange juice and frozenraspberries, and other food products. Fecal- or vomitus-contaminatedfood or water is a major source of infection. Other risk factors caninclude having an impaired immune system, living with a child whoattends a school or child care center, or being an infant or elderlyindividual.

Such methods can comprise contacting a first clinical sample from apatient, obtained prior to treatment with the therapeutic agent, with anorovirus-binding aptamer; detecting the presence of thenorovirus-binding aptamer bound to norovirus in the clinical sample;contacting a second clinical sample from the patient, obtained followingtreatment with the therapeutic agent, with the norovirus-bindingaptamer; detecting the presence of the norovirus-binding aptamer boundto norovirus in the second clinical sample; and then comparing thepresence of the norovirus-binding aptamer bound to norovirus in thefirst and second clinical samples. Decreased binding in the secondclinical sample relative to the first clinical sample can indicate thatthe therapeutic agent is effective in treating norovirus infection inthe patient.

Such methods can be used in the context of a clinical setting todetermine if a patient is responding to an approved therapeutic agentfor norovirus infection or in a preclinical or clinical trial setting toassess whether a candidate therapeutic agent is therapeuticallyeffective in treating norovirus infections.

In some cases, mixtures of norovirus-binding aptamers are usedcomprising a first norovirus-binding aptamer and at least one differentnorovirus-binding aptamer. In some cases, the first norovirus-bindingaptamer and the one or more other norovirus-binding aptamers bind todifferent epitopes on the same norovirus genogroup, genotype, or strain.In some cases, the first norovirus-binding aptamer and the one or moreother norovirus-binding aptamers preferentially bind to differentnorovirus genogroups, genotypes, or strains. In some cases, the firstnorovirus-binding aptamer and the one or more other norovirus-bindingaptamers are differentially labeled.

A clinical sample from a patient diagnosed with norovirus infection canfirst be evaluated to establish a baseline for the binding of one ormore norovirus-binding aptamers disclosed herein in the sample (i.e., abaseline for the presence of norovirus in the sample) before commencingtherapy with the therapeutic agent. In some instances, multiple clinicalsamples from the patient are evaluated to establish both a baseline andmeasure of random variation independent of treatment. A therapeuticagent can then be administered in a regime. The regime may includemultiple administrations of the therapeutic agent over a period of time.Optionally, binding of the aptamers (i.e., presence of norovirus) isevaluated on multiple occasions in multiple clinical samples from thepatient, both to establish a measure of random variation and to show atrend in response to therapy. The various assessments of aptamer bindingto the clinical samples can then be compared. If only two assessmentsare made, a direct comparison can be made between the two assessments todetermine whether aptamer binding (i.e., presence of norovirus) hasincreased, decreased, or remained the same between the two assessments.If more than two measurements are made, the measurements can be analyzedas a time course starting before treatment with the therapeutic agentand proceeding through the course of therapy. In patients for whomaptamer binding in clinical samples has decreased (i.e., theconcentration of norovirus has decreased), it can be concluded that thetherapeutic agent was effective in treating the norovirus infection inthe patient. The decrease in aptamer binding can be statisticallysignificant. Optionally, binding decreases by at least about 1%, atleast about 2%, at least about 3%, at least about 4%, at least about 5%,at least about 10%, at least about 15%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, or 100%.

Assessment of aptamer binding can be made in conjunction with assessingother symptoms of norovirus infection. The term “symptom” refers to asubjective evidence of a disease as perceived by a patient or objectiveevidence of a disease as observed by a physician. Relevant improvementsor deteriorations in symptoms of norovirus infection can be those whichin a physician's judgment are more likely than not due to the treatmentrather than random variation in the patient's condition or the infectionhaving run its natural course. Symptoms of norovirus infection includenausea, acute onset vomiting, watery non-bloody diarrhea, abdominalcramps, dehydration, low-grade fever, myalgia, malaise, muscle pain, andheadache. Onset of such symptoms typically occurs about 12-48 hourspost-exposure, and such symptoms typically last for about 18-72 hours.

The binding of aptamers (i.e., presence of norovirus) can be evaluatedin multiple patients, some who have been treated with the therapeuticagent and some who have not, to establish the efficacy of thetherapeutic agent in a population of patients. Such a population can besufficiently large as to include at least one patient in whose clinicalsamples aptamer binding decreases in response to treatment with thetherapeutic agent and at least one individual in whose clinical samplesaptamer binding increases or remains the same after treatment.Optionally, the population includes at least about 2, at least about 5,at least about 10, at least about 50, at least about 100, or at leastabout 1000 subjects. Optionally, binding of the aptamers (i.e., presenceof norovirus) is evaluated on multiple occasions in multiple clinicalsamples from multiple patients, some who have been treated with thetherapeutic agent and some who have not, both to establish a measure ofvariation in a population and to establish on average how long thenorovirus infection takes to run its course with and without thetherapeutic agent.

Depending on the outcome of the comparison of aptamer binding (i.e.,presence of norovirus) before treatment with the therapeutic agent andafter treatment, different patients may receive different subsequenttreatment regimes. In patients in whose clinical samples the aptamerbinding has decreased, it can be concluded that the first regime wassuccessful. The decrease in aptamer binding can be statisticallysignificant. Optionally, binding decreases by at least about 1%, atleast about 2%, at least about 3%, at least about 4%, at least about 5%,at least about 10%, at least about 15%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, or 100%.Such subjects can thereafter receive a second regime, which can be thesame as the first regime (because it was successful) or can involvecontinued administration of the same therapeutic agent as in the firstregime but at reduced dosage and/or frequency, or no further treatment.For example, in the second regime the dosage and/or frequency can bereduced by a factor of at least about 1.5, at least about 2, or at leastabout 1.5-5. In patients in whose clinical samples aptamer binding hasremained the same or increased, it can be concluded that the firstregime was unsuccessful or at least less than optimally successful. Suchsubjects thereafter can receive a second regime involving administeringthe same therapeutic agent as in the first regime but at an increaseddosage and/or frequency. For example, the dosage and/or frequency can beincreased by a factor of at least about 1.5, at least about 2, or atleast about 1.5-5. The second regime can also include administering adifferent therapeutic agent than in the first regime.

The instant disclosure further provides methods of evaluating theefficacy of a norovirus vaccine in one or more subjects. The term“subject” includes humans or mammals.

Such methods can comprise challenging one or more vaccinated subjectsand one or more non-vaccinated subjects with norovirus; contacting afirst set of one or more clinical samples from the one or morevaccinated subjects with a norovirus-binding aptamer; detecting thepresence of the norovirus-binding aptamer bound to norovirus in theclinical samples; contacting a second set of clinical samples from theone or more non-vaccinated subjects with a norovirus-binding aptamer;detecting the presence of the norovirus-binding aptamer bound tonorovirus in the clinical samples; and then comparing the presence ofthe norovirus-binding aptamer bound to norovirus in the first and secondsets of clinical samples. Decreased binding in the first set of clinicalsamples relative to the second set of clinical samples can indicate thatthe vaccine is effective in preventing or reducing norovirus infection.

The population of subjects can include at least about 2, at least about5, at least about 10, at least about 50, at least about 100, or at leastabout 1000 subjects, some who have received the vaccine and some whohave not. Optionally, binding of the aptamers (i.e., presence ofnorovirus) is evaluated on multiple occasions in multiple clinicalsamples from multiple subjects, some who have been vaccinated and somewho have not, to establish a measure of variation in a population.

In some cases, mixtures of norovirus-binding aptamers are usedcomprising a first norovirus-binding aptamer and at least one differentnorovirus-binding aptamer. In some cases, the first norovirus-bindingaptamer and the one or more other norovirus-binding aptamers bind todifferent epitopes on the same norovirus genogroup, genotype, or strain.In some cases, the first norovirus-binding aptamer and the one or moreother norovirus-binding aptamers preferentially bind to differentnorovirus genogroups, genotypes, or strains. In some cases, the firstnorovirus-binding aptamer and the one or more other norovirus-bindingaptamers are differentially labeled.

In some cases, clinical samples from the subjects can be evaluated priorto challenge with norovirus to establish a baseline for the binding ofone or more norovirus-binding aptamers disclosed herein in the samples(i.e., a baseline for the presence of norovirus in the sample) beforecommencing challenge with norovirus. In some instances, multipleclinical samples from the subjects are evaluated to establish both abaseline and a measure of random variation independent of treatment. Thesubjects can then be challenged with norovirus. Optionally, binding ofthe aptamers (i.e., presence of norovirus) is evaluated on multipleoccasions in multiple clinical samples from the subjects, both toestablish a measure of random variation and to show a trend in responseto challenge with norovirus. If more than two measurements are made, themeasurements can be analyzed as a time course starting before challengewith norovirus proceeding through the challenge with norovirus andpost-symptomatically. If aptamer binding in clinical samples is lower(i.e., the concentration of norovirus is lower) in vaccinated subjectspost-norovirus challenge compared to non-vaccinated subjectspost-norovirus challenge, it can be concluded that the vaccine waseffective in preventing or reducing norovirus infection. The differencein aptamer binding between clinical samples from vaccinated subjects andclinical samples from non-vaccinated subjects can be statisticallysignificant. Optionally, binding is at least about 1%, at least about2%, at least about 3%, at least about 4%, at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or 100% lower inclinical samples from vaccinated subjects than in clinical samples fromnon-vaccinated subjects.

Assessment of aptamer binding can be made in conjunction with assessingother symptoms of norovirus infection. Relevant symptoms can include,for example, nausea, acute onset vomiting, watery non-bloody diarrhea,abdominal cramps, dehydration, low-grade fever, myalgia, malaise, musclepain, and/or headaches.

VII. Kits

Also provided are kits including a norovirus-binding aptamer disclosedherein and instructions for use. Such kits can be used for, e.g.,performing the detection and diagnostic methods described above. A kitcan also include a label. Kits also typically contain labeling providingdirections for use of the kit. Labeling generally refers to any writtenor recorded material that is attached to, or otherwise accompanies, akit at any time during its manufacture, transport, sale or use. Forexample, the term labeling encompasses advertising leaflets andbrochures, packaging materials, instructions, audio or video cassettes,computer discs, as well as writing imprinted directly on kits. Such kitsmay also provide a positive control, for example, a norovirusvirus-like-particle in suspension, viral RNA, or a surrogate virus to beused as a process control. Such kits may further provide a solid supportto which the aptamers can be tethered, such as magnetic beads. Such kitsmay further provide primers and probes targeting a region of the genomeof at least one norovirus strain or targeting the aptamer itself.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited. Forexample, a composition that “comprises” or “includes” an aptamer maycontain the aptamer alone or in combination with other ingredients.

Designation of a range of values includes all integers within ordefining the range, and all subranges defined by integers within therange.

Unless otherwise apparent from the context, the term “about,” whenreferring to a value, is meant to encompass variations of +/−50%,+/−20%, +/−10, +/−5%, +/−1%, +/−0.5%, or +/−0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethods or employ the disclosed compositions.

The singular forms of the articles “a,” “an,” and “the” include pluralreferences unless the context clearly indicates otherwise. For example,the term “an aptamer” or “at least one aptamer” can include a pluralityof aptamers, including mixtures thereof.

All patent filings, website, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

TABLE 1 Brief Description of the Sequences SEQ ID NO Description 1 SMVaptamer 2 SMV aptamer 3 SMV aptamer 4 SMV aptamer 5 SMV aptamer: SMV-5(S-7) 6 SMV aptamer: SMV-18 7 SMV aptamer: SMV-22 (S-7) 8 SMV aptamer 9SMV aptamer 10 SMV aptamer 11 SMV aptamer 12 SMV aptamer: SMV-5 (S-9) 13SMV aptamer 14 SMV aptamer 15 SMV aptamer 16 SMV aptamer 17 SMV aptamer18 SMV aptamer 19 SMV aptamer 20 SMV aptamer 21 SMV aptamer 22 SMVaptamer 23 SMV aptamer: SMV-17 24 SMV aptamer 25 SMV aptamer: SMV-19 26SMV aptamer 27 SMV aptamer: SMV-21 28 SMV aptamer: SMV-22 (S-9) 29 SMVaptamer 30 SMV aptamer: SMV-25 31 SMV aptamer: SMV-26 32 SMV aptamer 33SMV aptamer 34 SMV aptamer 35 GII.4 P Aptamer: M1 36 GII.4 P Aptamer:M9-2 37 GII.4 P Aptamer: M12-2 38 GII.4 P Aptamer: M13-2 39 GII.4 PAptamer: M6-2 40 GII.4 P Aptamer: M5 41 NV Aptamer: NV 1-1 42 NVAptamer: NV 1-15 43 NV Aptamer 44 NV Aptamer 45 NV Aptamer 46 NV Aptamer47 NV Aptamer 48 NV Aptamer 49 NV Aptamer 50 NV Aptamer 51 NV Aptamer 52NV Aptamer 53 NV Aptamer: NV 1-24 54 NV Aptamer 55 NV Aptamer: NV 2-9 56NV Aptamer: NV 2-3 57 NV Aptamer 58 NV Aptamer 59 NV Aptamer 60 NVAptamer 61 NV Aptamer 62 NV Aptamer: NV 2-1 63 NV Aptamer 64 NV Aptamer65 NV Aptamer 66 NV VPg Aptamer: N6 67 NV VPg Aptamer: N3 68 NV VPgAptamer: N1 69 NV VPg Aptamer: N14 70 NV VPg Aptamer: N1-2 71 NV VPgAptamer: N4-2 72 NV VPg Aptamer: N11-2 73 NV VPg Aptamer: N12-2 74 TVVPg Aptamer: T5 75 TV VPg Aptamer: T9 76 TV VPg Aptamer: T1-2 77 TV VPgAptamer: T9-2 78 TV VPg Aptamer: T10-2 79 SMV-19 with constant regions80 SMV-21 with constant regions 81 SMV-25 with constant regions 82SMV-26 with constant regions 83 M-1 with constant regions 84 M6-2 withconstant regions 85 NV 2-1 with constant regions 86 NV 2-9 with constantregions 87 NV 2-3 with constant regions 88 NV 1-1 with constant regions89 NV 1-24 with constant regions 90 NV 1-15 with constant regions 91SMV-5 (S-7) with constant regions 92 SMV-18 with constant regions 93SMV-22 (S-7) with constant regions 94 SMV-5 (S-9) with constant regions95 SMV-22 (S-9) with constant regions 96 SMV-17 with constant regions 97M5 with constant regions 98 Motif 1 99 Motif 2 100 Motif 3 101 Motif 4102 Motif 5 103 Motif 6 104 Motif 7 105 Motif 8 106 Motif 9 107 Motif 10108 Motif 11 109 Motif 12 110 Motif 13 111 Motif 14 112 Motif 1, version1 113 Motif 1, version 2 114 Motif 1, version 3 115 Motif 2, version 1116 Motif 2, version 2 117 Motif 2, version 3 118 Motif 2, version 4 119Motif 3, version 1 120 Motif 3, version 2 121 Motif 4, version 1 122Motif 4, version 2 123 Motif 5, version 1 124 Motif 5, version 2 125Motif 6, version 1 126 Motif 6, version 2 127 Motif 7, version 1 128Motif 7, version 2 129 Motif 8, version 1 130 Motif 8, version 2 131Motif 9, version 1 132 Motif 9, version 2 133 Motif 10, version 1 134Motif 10, version 2 135 Motif 10, version 3 136 Motif 11, version 1 137Motif 11, version 2 138 Motif 12, version 1 139 Motif 12, version 2 140Motif 13, version 1 141 Motif 13, version 2 142 Motif 13, version 3 143Motif 14, version 1 144 Motif 14, version 2 145 Motif 14, version 3 146Generic combinatorial DNA library sequence 147 Forward constant regionprimer 148 Reverse constant region primer 149 JJV2F primer 150 COG2Rprimer 151 RING2-TP probe 152 GII4 P domain forward 153 GII4 P domainreverse 154 T7GII.4F 155 GII.4R2 156 P493 157 P494 158 Norwalk VPgforward 159 Norwalk VPg reverse 160 Tulane VPg forward 161 Tulane VPgreverse 162 Motif 1, version 4 163 Motif 2, version 5 164 Motif 3,version 3 165 Motif 4, version 3 166 Motif 5, version 3 167 Motif 6,version 3 168 Motif 7, version 3 169 Motif 8, version 3 170 Motif 9,version 3 171 Motif 10, version 4 172 Motif 11, version 3 173 Motif 12,version 3 174 Motif 14, version 4 175 COG2R primer, version 2 176Aptamer AP1-GI 177 Aptamer AP2-GI 178 Aptamer AP3-GI 179 Aptamer AP4-GI180 Aptamer AP5-GI 181 Aptamer AP6-GI 182 Motif 15 183 Motif 16 184Motif 17 185 Motif 18 186 Motif 19 187 Motif 20 188 Motif 21 189 Motif22 190 Motif 23 191 Motif 19, version 1 192 Motif 19, version 2 193Motif 19, version 3 194 Motif 20, version 1 195 Motif 20, version 2 196Motif 20, version 3 197 Motif 21, version 1 198 Motif 21, version 2 199Motif 21, version 3

EXAMPLES Example 1: Selection, Characterization and Application ofNucleic Acid Aptamers for the Capture and Detection of Human NorovirusStrains Materials and Methods Viruses and Virus-Like Particles (VLPs)

Viruses.

Snow Mountain virus (SMV), the prototype genogroup II, genotype 2(GII.2) human norovirus (HuNoV) and the target for aptamer selection,and Norwalk (NV), the prototype genogroup I, genotypel HuNoV, wereobtained as stool specimens originating from a human challenge study.Pre-challenge stool samples confirmed (by RT-qPCR) as negative for HuNoVwere used for counter selection and as negative controls in somestudies. Additional fecal specimens associated with previously confirmedHuNoV outbreaks (representing strains GI.6, GII.1, GII.3, GII.4, GII.7and an untypable GII) were also used in detection assays. All stoolsamples were suspended 20% in phosphate buffered saline (PBS). In somecases, these suspensions were used without further purification,designated as crude suspensions. In other cases, the suspensions werepartially purified using chloroform extraction (Shin & Sobsey, WaterRes. 42:4562-4568 (2008)). Hepatitis A virus HM175 (cell cultureadapted) and poliovirus 1 were partially purified (chloroform extracted)from cell culture lysates and used in exclusivity studies. All virussuspensions were stored at −80° C. until use.

Virus-Like Particles (VLPs).

Self-assembled non-infectious Virus-Like Particles (VLPs), producedusing the recombinant expression of HuNoV capsid proteins, were used aspurified candidate proteins for the characterization of aptamer bindingaffinity. The VLP panel consisted of representatives of genogroup I(GI.1 (Norwalk virus), GI.4, GI.6, GI.7 and GI.8)) and genogroup II(GII.1, GII.2 (SMV), GII.3, GII.4 (Houston and Grimsby virus), GII.6,GII.7, GII.12 and GII.17) HuNoV. VLPs were stored at 4° C. until use.

Selection of Aptamers Using SELEX (Systematic Evolution of Ligands byEXponential Enrichment)

Preparation of the DNA Library.

An 81-base combinatorial DNA library consisting of 40-mer random regionsflanked by forward and reverse constant regions(5′-AGTATACGTATTACCTGCAGC-(N)₄₀—CGATATCTCGGAGATCTTGC-3′) (SEQ ID NO:146) was used. Preparation of the dsDNA library for SELEX was done inaccordance with the method of Dwivedi et al., Appl. Microbiol.Biotechnol. 87:2323-2334 (2010). Specifically, the library was amplifiedusing a fluorescein (FAM)-labeled forward constant region primer(5′-56-FAM/-AGTATACGTATTACCTGCAGC-3′) (SEQ ID NO: 147) and abiotinylated reverse constant region primer(5′-/5Bios/-GCAAGATCTCCGAGATATCG-3′) (SEQ ID NO: 148). Briefly, a 50 μlreaction master mix containing 5 μl of aptamer library (10 μM), 1×GOTAQbuffer (Promega Corp., Madison, Wis.), 0.2 mM GENEAMP dNTPmix (AppliedBiosystems, Foster City, Calif.), 5 U GOTAQ DNA polymerase (Promega),and 500 nM of each primer were amplified as follows: 95° C. for 5 min,followed by 30 cycles of 95° C. (1 min), 55° (1 min), and 72° C. (1min), and a final extension at 72° C. for 10 min, using a DNA Engine(PTC-200) Peltier Thermal Cycler-200 (MJ Research/Bio-Rad Laboratories,Hercules, Calif.). The labeled dsDNA library was conjugated toStreptavidin MAGNESPHERE paramagnetic particles (SA-PMPs) (Promega)according to manufacturer instructions and harvested with an MPC-Mmagnetic particle concentrator (Invitrogen Dynal AS, Oslo, Norway). Thelibrary magnetic bead conjugate was washed three times in 0.1×SSC. TheFAM-labeled ssDNA moieties were then separated from the immobilizedbiotinylated strands by alkaline denaturation using 50 μl of 0.15M NaOHfor 7 min at room temperature (RT), followed by magnetic separation ofthe beads. The supernatant obtained was mixed with 1 ml of nuclease freewater followed by filtration to remove residual NaOH using a MICROCONYM-30 filter device (Millipore, Billerica, Mass.). It was then purifiedby ethanol precipitation with resuspension in 50 μl of nuclease freewater. The presence of FAM-labeled DNA was confirmed using a fluorescentplate reader (Tecan Safire, Tecan Group Ltd., Männedorf, Switzerland)and its concentration determined using a NanoPhotometer Pearl (ImplenGMbH, Munich, Germany).

Preparation of the Target for SELEX.

The target for SELEX was produced by immobilizing SMV to antibody-beadconjugates. Briefly, M-280 Tosylactivated DYNABEADS (Invitrogen Dynal)were cross-linked to mouse monoclonal antibodies against SMV (Abeam,Cambridge, Mass.) as per manufacturer instructions. One hundred μl ofthe partially purified SMV stock (consisting of about 10⁵-10⁶ RT-qPCRamplifiable units/nil) was mixed with 10 μl of the antibody-beadconjugate suspended in 500 μl of binding buffer (consisting of phosphatebuffered saline (pH 7.1) supplemented with 100 mg/L CaCl₂, 100 mg/LMgCl₂ and 0.05% Tween 20) followed by RT incubation for 2 h. Afterwashing thrice with PBS supplemented with 0.05% Tween 20 (PBST), theconjugate was resuspended in 20 μl of PBS and used for SELEX.

SELEX.

An aliquot of 300-500 pmoles of FAM-ssDNA pool suspended in 500 μl ofbinding buffer was added to 10 μl immobilized SMV target followed bygentle rotation for 45 min at RT. Aptamer-bound SMV was recovered bymagnetic capture, washed thrice in PBST, and the aptamers eluted fromthe bead-bound virus particles with 200 μl of nuclease free waterfollowed by heating at 90° C. for 5 min. The resulting aptamer pool waspurified by ethanol precipitation, re-amplified using the labeledconstant region primers, and the FAM-ssDNA aptamers were recovered asdescribed above. This constituted one selection round and a total of upto nine such iterations of the selection process were performed. Toavoid non-specific amplification of the recovered aptamer pool,single-stranded binding protein (Promega) at a concentration of 0.1μg/μl was added to the PCR reactions and the pool was amplified usingthe appropriate annealing temperatures (obtained by running atemperature gradient within a range of 55° C. to 65° C. on the recoveredaptamer pool).

Counter-SELEX.

Two sequential counter-SELEX rounds were done after each round of SELEXto impart specificity to the aptamer candidates against (1) thecomponents of a 20% HuNoV-negative human stool suspension; and (2) thebead-antibody complex (without SMV). In counter-SELEX, exposure of theaptamer pools to the negative stool specimens or bead-antibody complexwas done as described above, but in this case, the aptamers bound to thecomplex were discarded, while the unbound aptamer pool (supernatant) wasrecovered. This was purified by ethanol precipitation, reamplified byPCR, and used in another round of SELEX.

Identification of Aptamer Sequences.

After the 4^(th), 7^(th), and 9^(th) rounds of sequential SELEX andcounter-SELEX, final PCR product was cleaned using the QIAquick PCRpurification kit (Qiagen, Valencia, Calif.) and ligated into a pCR2.1TOPO vector (Invitrogen, Carlsbad, Calif.) according to manufacturerinstructions. Products were electroporated into E. coli Top10 cells(Invitrogen) and transformants selected as white colonies on (LuriaBroth)-Xgal agar plates containing 50 μg/ml kanamycin. The selectedtransformants were grown overnight in Luria Broth with kanamycin (50μg/ml) and the plasmid DNA was extracted using the QlAprep spin plasmidminiprep kit (Qiagen). The plasmid DNA was sent to Genewiz (SouthPlainfield, N.J.) for sequencing. The unique aptamers were identified bysequence analysis.

Characterization of Aptamer Candidates

Dissociation Constant and Structural Analysis of Aptamers.

An ELISA-like assay (Enzyme-Linked Aptamer Sorbent Assay, or ELASA,described below) was used to determine equilibrium dissociationconstants (K_(d)) for the candidate aptamers (see, e.g., Friguet et al.,J Immunol. Methods 77:305-319 (1985); Fuch et al., J. Immunol. Methods188:197-208 (1995)). This was done using SMV VLPs (3 μg/ml) anddifferent concentrations (10 nM, 100 nM, 500 nM, 1 μM, 2 μM) of eachaptamer. To estimate K_(d), plots of the ratio between absorbance ofTest samples/absorbance of Negative controls (T/N ratios, Y axis) as afunction of the aptamer concentration (X axis) were generated using anon-interacting binding sites model in Sigma Plot (Jandel, San Rafael,Calif.). Common sequence motifs were identified using the online MEMEserver, which can be found at the website located at meme.sdsc.edu.Structural folding analyses of the selected aptamers were done using theDNA Mfold online server, which can be found at the website located atmfold.rna.albany.edu/?q=mfold/DNA-Folding-Form (Zuker, Nucleic AcidsRes. 31:3406-3415 (2003)).

Binding Affinity Studies Using Enzyme-Linked Aptamer Sorbent Assay(ELASA).

Binding affinity studies were performed with candidate aptamers and VLPsusing a protocol adapted from a previously reported ELISA-based antigendetection assay (Rogers et al., J. Clin. Microbiol. 51:1803-1808(2013)). In this case, the antibody was replaced with an aptamer and theresulting procedure termed Enzyme-Linked Aptamer Sorbent Assay (ELASA).For this assay, the selected aptamers were labeled with a 5′ biotinmoiety. One hundred μl of pure VLP suspension (3 μg/ml) was placed ineach well of a covered, flat-bottomed polystyrene 96-well plate (Costar3591, Fisher, Pittsburgh, Pa.) and incubated overnight at 4° C. Aftercoating the wells with VLPs, the plates were blocked with 200 μl of 5%skim milk in PBST containing non-related DNA sequences (i.e., L.monocytogenes primers hlyQF/R and L23 SQF/R) (Rodriguez-Lazaro et al.,FEMS Microbiol. Lett. 233:257-267 (2004)), followed by overnightincubation at 4° C. After washing three times with PBST, 100 μl ofbiotinylated aptamer (1 μM) was added to each well and the plate wasincubated for an hour at RT with gentle mixing. After removing excessaptamers, the plates were washed three times with PBST. One-hundred μlof ELISA-grade streptavidin-horse radish peroxidase conjugate (1 mg/ml,1:5000, Invitrogen) was added to the plate and incubated at RT for 15min. The unbound conjugate was removed and the plate was washed fivetimes with PBST before applying 100 μl of 3,3′5,5′tetramethylbenzidine(TMB) peroxidase substrate (KPL, Gaithersburg, Md.) to each well. Theplate was incubated for 5 min at RT after which 100 μl of 1M phosphoricacid was added to stop the reaction. Absorbance at 450 nm was recordedusing a microplate reader (Tecan Infiniti M200pro). Negative controlsconsisted of no VLPs. As per convention (Ebel et al., Emerg. Infect.Dis. 8:979-982 (2002)), binding affinity was interpreted based on theratio between the absorbance readings for the test samples (to whichlabeled aptamer had been added) versus those for the negative control(PBS alone), (T/N ratios). Ratios <2.0 were considered negative byconvention (Ebel et al., Emerg. Infect. Dis. 8:979-982 (2002)). Ratiosbetween 2.0 and 5.0; >5.0 and 10.0; and >10.0 were interpreted as low,medium or strong binding, respectively. Ratios obtained for the negativecontrol were in the range of 0.1-0.3.

To evaluate binding inclusivity, ELASA was performed using 1 μM (100 μl)of each candidate aptamer as applied to a panel of virus-like particles(VLPs) corresponding to genogroup I (GI.1 (Norwalk virus), GI.4, GI.6,GI.7 and GI.8)) and genogroup II (GII.1, GII.2 (SMV), GII.3, GII.4(Houston and Grimsby), GII.6, GII.7, GII.12 and GII.17) HuNoV.Exclusivity analyses were done by ELASA using hepatitis A virus (HAV)and poliovirus. In all cases, PBS and SMV-VLPs were included as negativeand positive controls, respectively.

Application of Aptamers for Detection of HuNoV in Clinical and FoodSamples

Detection of HuNoV in Outbreak-Derived Stool Samples.

The ELASA assay was used to assess the performance of select aptamercandidates for detection of HuNoV in outbreak-derived fecal suspensions.Specimens evaluated included those representing strains GI.1 (Norwalk),GI.6, GII.1, GII.2 (SMV), GII.3, GII.4, GII.7 and an untypable GII.Briefly, 100 μl of ten-fold serially diluted partially purified fecalsuspensions were used to coat plates followed by incubation overnight at4° C. After three washes with PBST, the ELASA assay was done asdescribed above. PBS alone and fecal suspensions derived from uninfectedindividuals were used as negative controls. Due to limited availabilityof SMV VLPs, we used GII.4 (Houston) VLPs, which were also highlyreactive to aptamer 25, as the positive control.

Detection of HuNoV in Artificially Contaminated Lettuce Samples Using aCombined Pre-Concentration-Aptamer Magnetic Capture (AMC)-RT-qPCR.

Three g of lettuce (about 3×3 cm square) was disinfected by UV light andinoculated with 200 μl of serially diluted crude GII.4 virus stocksuspension (due to limited availability of SMV) at inoculumconcentrations ranging from 1-5 log₁₀ RNA copies per lettuce sample. Theinoculum was allowed to dry for 30 min. Virus pre-concentration was doneusing a previously reported elution-concentration method (Leggitt &Jaykus, J. Food Prot. 63:1738-1744 (2000)). Briefly, the samples weremixed with 25 ml of 0.5 M glycine-0.14 M NaCl buffer (pH 9.0), placed insterile Whirl-Pak-filter bags (Nasco, Fort Atkinson, Wis.) and stomached(Stomacher 400 Circulator, Seward, Norfolk, UK) at 230 rpm for one min.The recovered filtrate (containing the eluted viruses) was adjusted to0.9M NaCl and supplemented with 1% bovine serum albumin (Sigma Aldrich,St. Louis, Mo.), after which 12% polyethylene glycol (PEG) MW 8,000(Sigma Aldrich) was added. After incubation for 2 h at 4° C., sampleswere centrifuged at 10,000×g for 20 min at 8° C. and the recoveredpellet was resuspended in 1 ml of PBST.

The resuspended pellet was incubated with 1 μM of biotinylated aptamerfor an hour at RT with rotation. This was followed by the addition of 10μl of Streptavidin (SA)-C1 magnetic beads (from 10 mg/ml stock solution)(Invitrogen Dynal AS) previously blocked overnight with 5% skim milk inPBST followed by incubation for 25 min at RT. The bead-aptamer-virusconjugates were recovered using the magnetic particle concentrator. Theconjugates were washed twice with PBST, suspended in 100 μl PBS, and theRNA extracted using the NUCLISENS EASYMAG automated system (bioMerieuxSA, Marcy l'Etoile, France) according to manufacturer's instructions.The viral RNA was eluted in 40 μl of proprietary elution buffer andstored at −80° C. until used for amplification.

RT-qPCR was carried out using the SUPERSCRIPT III PLATINUM One-StepRT-qPCR system (Invitrogen) according to manufacturer instructions.Briefly, 25 μl RT-qPCR reactions consisted of 12.5 μl of 2× reactionmix, 5.5 μl DNase-RNase free water, 200 nM of each primer (JJV2F(5′-CAAGAGTCAATGTTTAGGTGGATGAG-3′) (SEQ ID NO: 149) and COG2R(5′-TCGACGCCATCTTCATTCACA-3′) (SEQ ID NO: 150)), and probe RING2-TP(5′-56-FAM TGGGAGGGCGATCGCAATCT-3BHQ-3′) (SEQ ID NO: 151) (Jothikumar etal., Appl. Environ. Microbiol. 71:1870-1875 (2005); Kageyama et al., J.Clin. Microbiol. 41:1548-1557 (2003)), 0.5 μl of the enzyme mix(SUPERSCRIPT III RT/PLATINUM Taq Mix) and 5 μl of the RNA template.Amplification was done under the following conditions: 50° C. for 15min, 95° C. for 2 min followed by 45 cycles of 95° C. for 15 sec, 54° C.for 30 sec, and 72° C. for 30 sec in a SmartCycler (Cepheid, Sunnyvale,Calif.). The RNA copy number was extrapolated from a standard curvebased on Ct values obtained by RT-qPCR amplification of serially dilutedsynthetic RNA as previously reported (Escudero et al., J. Food Prot.75:927-935 (2012)). Capture efficiency, expressed as a percentage, wasestimated from the standard curve and calculated as the ratio of theextrapolated RNA copies (after capture and detection by RT-qPCR) to thetotal input RNA copies per sample, multiplied by 100 (Joshi et al., Mol.Cell. Probes 23:20-28 (2009)). Negative controls consisted of capture byblocked beads in the absence of aptamer.

Statistical Analysis

Data were expressed as mean±standard deviation of three replicates ofeach experiment. The data were analyzed by one-way analysis of variance(ANOVA) with the Tukey's multiple comparison test using GraphPad Prismver. 5.0 d (San Diego, Calif.) or by Student's t-test. Values of p<0.05were considered statistically significant.

ssDNA Aptamer Selection

After 4, 7, and 9 rounds of sequential SELEX and counter-SELEX, 34unique aptamer candidates were identified from a total of 80 clonessequenced. All sequences are provided in Table 2. Candidates designatedas SMV-19, SMV-21, SMV-25 and SMV-26 were selected for further analysisas they were the most abundant in the identified aptamer pool(identified between 5-10 times) and showed strong preliminary bindingaffinity for SMV, genogroup I.1 (Norwalk), and genogroup 11.4 (Houston)VLPs using the ELASA assay (data not shown). FIGS. 1-4 show thepredicted structural folding of aptamers SMV-19, SMV-21, SMV-25 andSMV-26, respectively, with the forward and reverse constant regionsincluded. Other aptamers identified included SMV-5 (S-7) (dG=−4.74Kcal/mol), SMV-18 (dG=−4.92 Kcal/mol), SMV-22 (S-7) (dG=−6.41 Kcal/mol),SMV-5 (S-9) (dG=−6.75 Kcal/mol), SMV-22 (S-9) (dG=−8.62 Kcal/mol), andSMV-17 (dG=−6.19 Kcal/mol). SMV-5 (S-7), SMV-18, and SMV-22 (S-7) havebinding affinities characterized by equilibrium dissociation constantvalues of 40 nM, 45 nM, and 142 nM, respectively, as determined byELASA. These are similar to K_(d) values (0.040 μM) obtained forantibodies against norovirus using the same methodology.

TABLE 2 Aptamer Sequences Obtained from 4^(th), 7^(th) and 9^(th)Rounds of SELEX for SMV Round of SEQ # Aptamer SELEXRandom Region Sequence ID NO Repeats Identifier 4TGTTGGATTTTACGAAAAACGTGCTTACTTCATAGCGGCC  1  1 4GGTTGGGTAAGGGGGTCTGGTCAGGTAGGGCGGGGGGGGG  2  1 4TCGTAAACCCCTTATCCGTGAACCTTCAGCGGTAGACGCT  3  2 4CTCCCTCCAGCCTGCCTATTTTGCTTGGTTACGCATCTGT  4  1 7CCAGCGAAGGAAAGTCTTGGTTGGTCTAGTTTTTCGTGTG  5  2 SMV-5 (S-7) 7CTACGTGTGCGTTCCGATTGTTTAAATTGCTCAATGTATG  6  2 SMV-18 7CACACCACCTGAATTCCAGCACACTGGCGGCCGTTACTAG  7  2 SMV-22 (S-7) 9CACTCGACCTTCAGGGCGGCTTCTCAGCGTGTAGTGGTGA  8  1 9CTCGACTGATAGACCTAGCGTCAATCCTCATTGTTCGCTG  9  1 9CCAGTATTAGAGTCCTACTTTACACCGCTCTTGGCATCGT 10  1 9CACATGATAAGGTCGCGTGACTGTGAGTTAGTTGTTACAC 11  2 9TCGGCATAGGTCAAGTCGCTTCATTTGGATTAAGTTGAGG 12  1 SMV-5 (S-9) 9CACATACCAAAGTATTGGTCGCTAACTTTCGCCCAATTGA 13  1 9CTACGAGGTGGTTATAAGAGAACTTATCCGTGTTGGTTGC 14  1 9TGGTAGTGGGATATAGTTTTTCCAAGCGTACCCAGTTCTG 15  2 9CTATCAGCCATGAATTGCATTACCTTTGTTCTCCCCTTGC 16  1 9CCCCTCGGAAGATAGATTTTGCGAGAGTCTTGGGTTGAGG 17  1 9CCAGATAGCAGCACCTAATCTTATCCCTTTTATTTTTGGT 18  2 9TCGGGGGGAGGAGGGGGAATGGGAAGAAGGAGGTCGAGGG 19  1 9TGGATTACACGGCTAACTTCCCTGGTTCTTTTCTTTGATG 20  2 9TGGACGTTATTTGCACTCGTCGAACCCTATCATGCCTCCT 21  1 9CCTCATGCACAAAGGCTTATTACGGTCTAATTCTTTATAA 22  1 9TCGACATTATGTTTGACATCGATTGTTAATGTTTCTTTGC 23  2 SMV-17 9CCCCTACACAGTAAAATTCTTTAACACCTAGATCTTCGAC 24  2 7, 9CACCAGTGTGTTGAGGTTTGAGCACACTGATAGAGTGTCA 25  9 SMV-19 9TGAGCCTCCGTTTTAGTGATCAGAAGGGATGTGTGGCGTA 26  1 9CCATGTTTTGTAGGTGTAATAGGTCATGTTAGGGTTTCTG 27  9 SMV-21 9CGAGGGATACATGCTGACTATGGAATTATTTGAATTCCCA 28  4 SMV-22 (S-9) 9CTACAGGAGTTCATCTGGGAGAGTGTAAAGGATGAGGTGG 29  2 7, 9CATCTGTGTGAAGACTATATGGCGCTCACATATTTCTTTC 30 10 SMV-25 9TGACCGAGTGTCTGGTCATTTTCGATGTCTGTTGTTAGGC 31  7 SMV-26 9CCCTCCTTATCTCTGCTAATGGTTGATCCGTGTCCCGTAC 32  1 9CCCTGTTATCCTTATCCAACGAGCTTAATGTAACTTGGAC 33  2 9TGGGGGAGTGGTAGGTGTGCTGTGAAGGGGAGGGTTGGGG 34  1Characterization of ssDNA Aptamer Candidates

The structural folding for SMV-19, SMV-21, SMV-25, and SMV-26demonstrated a dominant loop and two protruding hairpins (FIGS. 1-4).Three motifs were observed when comparing the random sequence regions ofthe four aptamers using the MEME program (see Bailey & Elkan, Proc. Int.Conf. Intell. Syst. Mol. Biol. 2:28-36 (1994)). The motifs areidentified in Tables 3 and 4 and in the boxes in FIGS. 1-4. In Table 3,motif 1 is bolded and underlined, motif 2 is underlined, and motif 3 isbolded. Motif 1 (TGNNAGNN; SEQ ID NO: 98) was found near the 3′ end ofthe aptamers and was found in aptamers SMV-19, SMV-21, and SMV-26. Motif2 (NNNTGTNNNG; SEQ ID NO: 99) was found in all four aptamers, and isinvolved in notable stem-loops in all four of the aptamers, which mayimply a conserved interaction site with human noroviruses. Motif 3(AGGTNT; SEQ ID NO: 100) was found in aptamers SMV-19 and SMV-21immediately downstream of motif 2, possibly as one larger motif betweenthe two with the exception of an insertion of a thymine residue inaptamer SMV-21. These motif 2-3 segments are involved in the formationof similar stem-loops in both of the aptamers (see FIGS. 1 and 2). Suchsecondary structures have been considered as possible putative bindingepitopes on aptamers (Kato et al., Nucleic Acids Res. 28:1963-1968(2000)), so these stem-loops may be involved in aptamer binding tonorovirus.

TABLE 3 Positions of Motifs 1, 2, and 3 in Aptamers SMV-19,SMV-21, SMV-25, and SMV-26 Aptamer Identifier Sequence SEQ ID NO SMV-19CACCAGTGTGTTG AGGTTTGAGCACAC TGATAGAG TGTCA 25 SMV-21CCATGTTTTGTAGGTGTAATAGGTCA TGTTAGGG TTTCTG 27 SMV-25CATCTGTGTGAAGACTATATGGCGCTCACATATTTCTTTC 30 SMV-26TGACCGAGTGTCTGGTCATTTTCGATGTCTGT TGTTAGGC 31

TABLE 4 Motifs 1, 2, and 3 in Aptamers SMV-19,SMV-21, SMV-25, and SMV-26 Aptamer Identifier Motif 1 Motif 2 Motif 3SMV-19 TGATAGAG CAGTGTGTTG AGGTTT (SEQ ID NO: 112) (SEQ ID NO: 115)(SEQ ID NO: 119) SMV-21 TGTTAGGG CCATGTTTTG AGGTGT (SEQ ID NO: 113)(SEQ ID NO: 116) (SEQ ID NO: 120) SMV-25 n/a CTGTGTGAAG n/a(SEQ ID NO: 117) SMV-26 TGTTAGGC GAGTGTCTGG n/a (SEQ ID NO: 114)(SEQ ID NO: 118)

Equilibrium dissociation constant (K_(d)) values approximated for theaptamers were 191 nM for aptamer SMV-19; 101 nM for SMV-21; 232 nM forSMV-25; and 281 nM for SMV-26. FIG. 5A-D shows the correspondingequilibrium dissociation (K_(d)) curves for SMV-19, SMV-21, SMV-25, andSMV-26, respectively, generated using the one site binding model. Theequilibrium dissociation curves were generated by ELASA with SMV VLPs (3μg/ml) and various concentrations of aptamer (10 nM, 100 nM, 500 nM, 1μM, and 2 μM). To estimate K_(d), plots of the T/N ratios (absorbance at450 nm) as a function of the aptamer concentration were fitted to anon-interacting binding sites model with the equation Y=Bmax X/Kd+X “T”represents absorbance readings for the test sample, and “N” representsabsorbance readings for the negative control. The regressioncoefficients (R²) associated with this model ranged from 0.95 to 0.99.

Signal intensity ratios (T/N) in ELASA as evaluated for aptamers SMV-19,SMV-21, SMV-25 and SMV-26 using a panel of VLPs ranged from a low of 1.3to a high of 18.1 (Table 5). T/N values in Table 5 indicate the ratio ofabsorbance readings for the test sample (T) versus negative control (N)using ELASA. Per convention (Ebel et al., Emerg. Infect. Dis. 8:979-982(2002)), results less than 2.0 were considered negative (−). Low (+/−),medium (+) or strong (++) binding were interpreted for ratios between 2and 5; >5 and 10; and >10, respectively. All experiments were done intriplicate. Aptamer SMV-21 demonstrated medium to high binding affinitywith VLPs corresponding to GI.7, GII.1, GII.2, GII.3, GII.4 (both VLPs),GII.7, GII.12, and GII.17 (T/N ratios ranging from 6.4 to 18.1). AptamerSMV-25 reacted positively with VLPs corresponding to GI.4, GI.8, GII.1,GII.2, GII.3, GII.4 (both VLPs), GII.6, and GII.7 (T/N ratios rangedfrom 5.4 to 12.4). Aptamers SMV-19 and SMV-26 were less broadlyreactive. In general, T/N ratios were higher for GII VLPs than for GIVLPs. Although all four aptamers showed variable binding affinity todifferent genotypes in genogroups I and II, binding affinity was highestfor the GII.2 VLP, which is represented by SMV, the target used for theaptamer selection. Binding affinity was also quite high for GII.4Houston VLPs. Relative to exclusivity analysis, binding affinity (T/N)of the four aptamers to the non-target virus poliovirus was in the rangeof 3.2-4.4, and for HAV, from 2.8-3.4. These values were statisticallysignificantly lower (p<0.05) as compared to the positive control (GII.2(SMV) VLPs) (see FIG. 8 and above).

TABLE 5 Binding Affinity of Selected Aptamers Against a Broad Panel ofHuNoV VLPs Aptamers VLPs SMV-19 SMV-21 SMV-25 SMV-26 Genogroup T/NBinding T/N Binding T/N Binding T/N Binding GI.1 5.4 ± 0.9 (+) 2.4 ± 0.1(+/−) 3.0 ± 0.4 (+/−) 2.6 ± 0.5 (+/−) (Norwalk) GI.4 1.7 ± 0.3 (−) 4.3 ±0.2 (+/−) 5.6 ± 0.2 (+) 1.1 ± 0.2 (−) GI.6 3.7 ± 1.3 (+/−) 3.8 ± 0.1(+/−) 2.4 ± 0.1 (+/−) 1.5 ± 0.4 (−) GI.7 10.3 ± 0.6  (++) 8.1 ± 0.1 (+)3.5 ± 0.4 (+/−) 1.8 ± 0.8 (−) GI.8 1.7 ± 0.2 (−) 4.6 ± 0.3 (+/−) 6.2 ±0.1 (+) 1.2 ± 0.1 (−) GII.1 7.1 ± 0.3 (+) 8.6 ± 0.3 (+) 9.4 ± 0.1 (+)2.1 ± 0.3 (+/−) GII.2 (SMV) 12.9 ± 5.1  (++) 18.1 ± 3.2  (++) 12.4 ±1.1  (++) 4.1 ± 0.8 (+/−) GII.3 1.7 ± 0.9 (−) 11.8 ± 2.7  (++) 5.4 ± 0.1(+) 2.4 ± 0.5 (+/−) GII.4 9.6 ± 4.8 (+) 6.4 ± 1.9 (+) 10.4 ± 0.8  (++)3.2 ± 0.3 (+/−) (Grimsby) GII.4 12.5 ± 4.2  (++) 11.3 ± 1.2  (++) 11.0 ±1.1  (++) 2.8 ± 0.4 (+/−) (Houston ) GII.6 1.8 ± 1.0 (−) 3.0 ± 0.2 (+/−)7.3 ± 0.7 (+) 2.8 ± 0.3 (+/−) GII.7 4.0 ± 1.9 (+/−) 13.5 ± 1.4  (++) 9.1± 2.1 (+) 2.8 ± 0.8 (+/−) GII.12 1.5 ± 0.4 (−) 6.6 ± 0.1 (+) 1.9 ± 0.1(−) 2.4 ± 0.5 (+/−) GII.17 3.0 ± 1.6 (+/−) 12.2 ± 0.3  (++) 2.0 ± 0.1(+/−) 1.3 ± 0.1 (−)

Despite the targeting of a single virus (SMV) in SELEX, two of theidentified aptamers (candidates SMV-21 and SMV-25) showed bindingaffinity to a panel of HuNoV VLPs, demonstrating the efficacy of SELEXin identifying aptamers with binding inclusivity to HuNoV strains. Thebinding inclusivity of aptamer SMV-25 was further confirmed by thedetection of HuNoV strains in outbreak-derived fecal samples by ELASA(see above). The performance of aptamer SMV-25 with outbreak stoolspecimens containing GII.1, GII.2, GII.3, and GII.4 HuNoV is consistentwith the VLP binding data, all of which gave positive signals withELASA. Likewise, poor performance of aptamer SMV-25 with an outbreakspecimen corresponding to GI.6 is consistent with the low degree ofbinding to that VLP. Binding of stool specimens containing GI.1 viruswas less consistent with VLP data, as was lack of binding to GII.7specimens. Such inconsistency between VLP binding assays and applicationto outbreak-derived stool specimens could be a function of residualmatrix-associated aptamer binding (or interference with aptamer binding)or potential differences between the behavior of VLPs and native virus,which has been discussed amongst experts in the field.

In general, the ELASA T/N ratios were higher for GII strains than for GIstrains. This is not unexpected as major capsid protein (VP1) amino acidsequences for GII strains differ from those for GI strains by over 61.4%(Zheng et al., Virology 346:312-323 (2006)), and our SELEX target (SMV)was a GII strain. Nonetheless, different VLP binding patterns for thefour characterized aptamers indicate that they may bind to slightlydifferent regions of the viral capsid. The more broadly reactiveaptamers could also bind to more highly conserved areas of the virus,such as the shell or P2 domains (Lindesmith et al., PLoS Med 5:e31(2008)). Of the three common motifs identified in this study, motif 2was found in all four aptamers, and this may imply a conservedinteraction site with HuNoV. Secondary structures have been consideredas possible putative binding epitopes on aptamers (Kato et al., NucleicAcids Res. 28:1963-1968 (2000)).

Antibodies, the most frequently used ligands for HuNoV capture anddetection (Yao et al., Lett. Appl. Microbiol. 49:173-178 (2009); Park etal., Appl. Environ. Microbiol. 74:4226-4230 (2008); Lee et al., J. FoodProt. 76:707-711 (2013)), tend to lack broad reactivity, meaning thatsubsequent assays developed with these antibodies lack analyticalsensitivity (Costantini et al., J. Clin. Microbiol. 48:2770-2778(2010)). Having an alternative ligand type showing broad reactivity tomultiple HuNoV strains provides another tool upon which capture anddetection assays may be based. So, for example, aptamers SMV-19, SMV-21,and SMV-25 could be used as a polyvalent cocktail to impart a high levelof inclusivity for HuNoV capture and detection. They could also be usedin combination with other ligands such as antibodies or peptides (Rogerset al., J. Clin. Microbiol. 51:1803-1808 (2013)).

Application of Aptamers for Capture and Detection of HuNoV in HumanStool and Lettuce Samples

ELASA assays using aptamer SMV-25 were performed on serially dilutedpartially purified outbreak-derived stool specimens. The 10-20% stoolsuspensions were chloroform-extracted, diluted, and tested using ELASA.Experiments were done in triplicate. Statistically significantdifferences between the ratios obtained from the virus-containing stoolspecimens and the NVF (HuNoV-negative human stool suspensions) aredesignated with an asterisk (p<0.05). Results are expressed as ratiosbetween absorbance readings for test sample versus negative control(T/N). The T/N ratios corresponding to GI.1 (Norwalk), GII.1, GII.2(SMV), GII.3, GII.4, and GII untypable outbreak specimens were allstatistically significantly higher (p<0.05) when compared to the ratiosobtained for either negative control samples (i.e., HuNoV-negative humanstool suspensions (NVF) and no aptamer controls (PBS alone)) (FIG. 6).T/N ratios were higher for GII.2 (SMV), GII.1 and GII.4 outbreakspecimens relative to GI.1 (Norwalk), GII.3, and GII untypable. PositiveELASA signals were not obtained for outbreak specimens corresponding toGI.6 and GII.7 HuNoV strains (data not shown).

AMC-RT-qPCR using aptamer SMV-25 was performed on 3 g lettuce samplesartificially inoculated with different concentrations of GII.4 fecalstock. The negative controls consisted of the samples containing blockedbeads in the absence of the aptamer. Experiments were done intriplicate. Statistically significant differences are designated with anasterisk (p<0.05). When virus on artificially contaminated lettucesamples (inoculated with GII.4 at levels ranging from 1-5 log₁₀ RNAcopies per sample) were pre-concentrated, used in AMC with aptamerSMV-25, and detected after RNA extraction using RT-qPCR, a detectionlimit of about 1 log₁₀ RNA copies per 3 g lettuce was obtained (FIG. 7).Over this inoculum range, the capture efficiency of the combinedpre-concentration-AMC-RT-qPCR assay ranged from 2.5-36%. Captureefficiency (CE) was significantly higher (p<0.05) than the negativecontrols which consisted of blocked beads in the absence of aptamer.Capture efficiency increased with decreasing virus concentration.

Using the ELASA assay, we were able to approximate the SMV aptamer K_(d)values to be in the 100-200 nM range (see above). This is similar tomost commercial antibodies, which have K_(d) values in the low μM to nMrange (examples can be found at the website located at www.Abcam.com).In binding studies specifically with Norwalk virus VLPs, monoclonalantibodies have been shown to have K_(d) values in the low nM range(Chen et al., J. Virol. 87:9547-9557 (2013)), and K_(d) values forenteric virus binding protein as applied to HuNoV VLPs was similarly inthe range of 210-240 nM (Imai et al., BMC Biotechnol. 11:123 (2011)).Using dilution series experiments (data not shown), we found thedetection limits of the ELASA method to be about 2 log₁₀ genomic copiesbetter than commercial enzyme immunoassays that have previously beenapplied to fecal samples (Costantini et al., J. Clin. Microbiol.48:2770-2778 (2010); Kele et al., Diagn. Microbiol. Infect. Dis.70:475-478 (2011)).

Aptamer SMV-25 performed quite well when applied to a virus concentratederived from an artificially contaminated model food product using acombined virus pre-concentration followed by AMC-qPCR assay format. Useof aptamers for pre-concentration of microbes has been reported recentlyby others (Ozalp et al., Anal. Biochem. 447:119-125 (2014)).Comparatively speaking, the detection limit and capture efficiency ofthis method, at 10 RNA copies and 36%, respectively, were comparable tothose for HuNoV immunomagnetic separation-RT-qPCR assays applied toartificially contaminated fresh produce items (Park et al., Appl.Environ. Microbiol. 74:4226-4230 (2008); Lee et al., J. Food Prot.76:707-711 (2013)) and clinical specimens (Yao et al., Lett. Appl.Microbiol. 49:173-178 (2009)). Our detection limits were also similar ifnot better than those for other capture approaches that use morenon-specific ligands such as histo-blood group antigens (Tian et al.,Int. J. Food Microbiol. 147:223-227 (2011); Morton et al., Appl.Environ. Microbiol. 75:4641-4643 (2009); Pan et al., Food Microbiol.30:420-426 (2012)) and porcine gastric mucin (Tian et al., Appl.Environ. Microbiol. 74:4271-4276 (2008)).

Binding Exclusivity Analysis for Aptamers SMV-19, SMV-21, SMV-25, andSMV-26

Binding of aptamers SMV-19, SMV-21, SMV-25, and SMV-26 to hepatitis Avirus (HAV) and poliovirus was measured to analyze binding exclusivity.Virus suspensions (cell culture lysates) were chloroform-extracted priorto use in ELASA. SMV-VLPs were used as positive controls. The values ofabsorbance at 450 nm for the negative controls (PBS) were 0.1±0.045.Experiments were done in triplicate. Results are expressed as ratiosbetween absorbance readings for test sample versus negative control(T/N). Statistically significant differences are designated with anasterisk (p<0.05). The T/N ratios corresponding to SMV were allstatistically significantly higher (p<0.05) when compared to the ratiosobtained for either HAV or poliovirus, indicating that aptamers SMV-19,SMV-21, SMV-25, SMV-26 have little cross-reactivity with other viruses(FIG. 8).

Example 2: Capture and Detection of Norovirus by Target Specific NucleicAcid Aptamers Materials and Methods

Norovirus-Specific ssDNA Aptamers

The 81-mer ssDNA aptamer candidates (designated SMV-17 and SMV-22(S-9)), previously selected against SMV (genogroup 11.2 human NoVstrain) using a whole-virus SELEX approach were used in this study.These were selected from a larger aptamer candidate pool because oftheir apparent high binding affinities (K_(d)) and low free energies(dG) (Suh et al., Capture and Detection of a Representative HumanNorovirus Strain using Target-Specific Nucleic Acid Aptamers: Proof ofConcept, IAFP, Charlotte, N.C. (2013)), as previously evaluated using anEnzyme-linked Aptamer Sorbent assay (ELASA) (Bruno et al., J. Biomol.Tech. 22:27-36 (2011)) and the on-line software DNA Mfold version 3.2(found at the website located atmfold.bioinfo.rpi.edu/cgi-bin/dna-forml.cgi) (Zuker, Nucleic Acids Res.31:3406-3415 (2003)), respectively.

Prototype Assay Development

Two different approaches were used for aptamer-based capture anddetection of SMV. The first assay design, called aptamer magneticcapture (AMC), used ssDNA aptamers as capture ligands, with subsequentdetection using virus-specific RT-qPCR. In the second assay, asandwich-based format, SMV was captured using antibody-bound beads,which were then exposed to SMV-specific, ssDNA aptamer. Detection wasachieved by amplification of the SMV-specific aptamer using qPCR.

Method 1: Aptamer Magnetic Capture (AMC)-RT-qPCR.

One hundred μl of 10-fold serial dilutions of SMV (corresponding to 1-4log_(in) genome equivalent copies or GEC) were suspended in 1 ml PBS andexposed to a 2 μM solution of biotinylated aptamer SMV-17. Afterincubation at room temperature for 45 min, immobilization of thesuspended aptamer-bound SMV was done by exposure to 20 μl ofstreptavidin-coated magnetic beads (M-280, Invitrogen-Dynal AS, Oslo,Norway) previously blocked with SUPERBLOCK buffer (Thermo Scientific,Rockford, Ill.). The aptamer-bead-bound SMV complexes were washed twicewith 1×PBS and resuspended in 50 μl of DEPC-treated water.

The viral RNA was released by heat at 95° C. for 5 min prior toamplification by RT-qPCR using primers targeting the GII HuNoV ORF1/ORF2junction (JJV2F (5′-CAAGAGTCAATGTTTAGGTGGATGAG-3′) (SEQ ID NO: 149) andCOG2R (5′-GACGCCATCTTCATTCACA-3′) (SEQ ID NO: 175), and TAQMAN probeRing 2P (5′/56-FAM-TGGGAGGGCGATCGCAATCT-3/BHQ_1-3′) (SEQ ID NO: 151)) aspreviously described (Jothikumar et al., Appl. Environ. Microbiol.71:1870-1875 (2005); Kageyama et al., J. Clin. Microbiol. 41:1548-1557(2003)). Quantification of RNA copies was extrapolated from a standardcurve based on Ct values obtained by RT-qPCR amplification of seriallydiluted synthetic RNA (SMV genomic location 5003-5424) (Escudero et al.,J. Food Prot. 75:927-935 (2012)). The highest dilution providing aquantifiable RT-qPCR value was equated to one (1) Genome Equivalent Copy(GEC). The capture efficiency was expressed as the ratio of GEC detectedin the sample after aptamer capture and subsequent RT-qPCR, compared tothe input GEC concentration (all estimated using the standard curve),multiplied by 100.

Method 2: Two-Site Binding Sandwich qPCR (Sandwich Method).

Anti-HuNoV GII.2 antibody was conjugated to DYNABEADS M-280Tosylactivated (Invitrogen-Dynal) as per manufacturer instructions,followed by extensive washing and blocking with 0.5% bovine serumalbumin (Sigma-Aldrich, St. Louis, Mo.). A 50 μs aliquot ofantibody-conjugated beads was used to capture virus in one ml volumes of10-fold serially diluted SMV (corresponding to 1-4 log_(in) genomeequivalent copies or GEC). The mixture was incubated for 2 h at roomtemperature with gentle rotation, followed by subsequent washings andmagnetic pull-down to remove unbound or nonspecifically-bound viruses.The virus-bound beads were then blocked by the addition of a 5% skimmilk-PBS-Tween solution containing 10 μM of ssDNA 20-mers. Afterblocking overnight at 4° C. with gentle rotation, the beads were washedagain in PBS-Tween, concentrated by magnet, and supplemented with 10 nMof SMV-specific, ssDNA aptamer SMV-22 (S-9). The aptamer bindingreaction was carried out for 1 h at room temperature with rotation,after which the aptamer-virus-bead complexes were washed sequentiallywith PBS-Tween and resuspended in 50 μl of DEPC-treated water.

The ssDNA aptamers were the target for subsequent qPCR detection, whichwas done using the SYBR Green PCR Master Mix (Applied Biosystems,Warrington, UK) and primers targeting the constant regions of aptamerSMV-22 (S-9) (Forward: 5′-AGTATACGTATTACCTGCAGC-3′ (SEQ ID NO: 147),Reverse: 5′-GCAAGATCTCCGAGATATCG-3′) (SEQ ID NO: 148) (Dwivedi et al.,Appl. Microbiol. Biotechnol. 87:2323-2334 (2010)). Each reaction tubecontained 12.5 μl of 2×SYBR Green Master Mix (Applied Biosystems), 9.6μl of PCR-grade water, 0.2 μl of a 10 μM concentration of primer set,and 2.5 μl of template DNA in a 25 μl PCR mixture. The PCR amplificationwas performed in a SmartCycler using a two-step thermal protocol ofinitial denaturation at 95° C. for 10 min followed by 45 cycles ofdenaturation at 95° C. for 15 sec and annealing/extension at 60° C. for60 sec. Fluorescence signals were measured once per cycle at the end ofthe extension step. After PCR amplification, T_(m) curve analysis wasperformed by adding the analysis of fluorescence from 60° C. to 95° C.(0.2° C./sec).

The two-site binding sandwich assay was compared to immunomagneticseparation (IMS) without the subsequent aptamer binding step. Briefly,50 μg of magnetic beads to which anti-NoV GII.2 antibody was conjugatedwere exposed to serially diluted SMV. After capture, the samples werewashed, viral RNA released by heat, and downstream detection performedusing RT-qPCR targeting the virus ORF1-ORF2 junction.

Statistical Methods

One-way analysis of variance (ANOVA) with the Tukey's multiplecomparison test (p<0.05) was used to analyze the AMC assay data. For thetwo-site binding sandwich assay format and IMS, comparisons betweengroups were performed using t-tests. Values of p<0.05 were consideredstatistically significant. All statistical analyses were done usingGraphPad Prism ver. 5.0d (San Diego, Calif.).

Results and Discussion Aptamer Magnetic Capture (AMC)-RT-qPCR Method

The lower limit of detection (LoD) of the combined AMC-RT-qPCR assayusing aptamer SMV-17 was approximately 1 log₁₀ GEC SMV/1 ml (FIG. 9).The mean capture efficiency (%) of aptamer SMV-17 as applied to seriallydiluted SMV stock ranged from 2.5%-34%, and increased (improved) asvirus titer decreased (from 4 log₁₀-1 log₁₀ GEC/ml). The parallelIMS-RT-qPCR performed in this study showed limit of detection about 4log₁₀ GEC/ml with less than 2.5% of capture efficiency.

Two-Site Binding Sandwich qPCR Method

The relationship between initial SMV concentration (prior to theantibody capture step) and the Ct values obtained after amplification ofaptamer SMV-22 (S-9) sequence is shown in FIG. 10. The x-axis representsthe initial input of SMV, and the y-axis represents the qPCR Ct value(mean±standard deviation). Experiments were done by triplicate. Anasterisk designates statistical significance (p<0.05) when comparing theCt values of samples containing SMV to that of the negative controlwhich did not contain SMV. As expected, the Ct values of samplescontaining high concentrations of SMV (>4 log₁₀ GEC) were very low(<18), and these Ct values gradually increased as the concentration ofSMV decreased. The negative control sample (no SMV exposure duringantibody capture but exposure to the aptamer) had a Ct value ofapproximately 21.5, indicating a relatively high degree of non-specificbinding of the aptamer to the antibody-coated magnetic beads. In casessuch as these, assay positivity is usually established at values >2-3times the standard deviation associated with the negative controlsamples. This criteria was met for samples containing between 1 log₁₀and 4 log₁₀ GEC SMV/1 ml, equating to a lower limit of detection of 1log₁₀ (10) GEC SMV.

To better understand the dynamics of the antibody capture step of thetwo-site binding sandwich qPCR method, simple IMS/RT-qPCR assaystargeting the viral genome were performed (FIG. 11). In this case, thex-axis represents the initial input log₁₀ GEC of SMV, and the y-axisrepresents the Ct value as a result of RT-qPCR (mean±standard deviation)after antibody capture. For statistical comparison purposes, samples(1-3 log₁₀ GEC) that were non-detectable by RT-qPCR were designated witha Ct value of 45. Experiments were done by triplicate. An asteriskdesignates statistical significance (p<0.05) when comparing the Ctvalues of samples containing SMV to that of the negative control whichdid not contain SMV. As expected, Ct values were much higher when theinitial SMV concentration was 4 log₁₀ GEC (Ct approx. 28). This alsoconstituted the limit of detection of the assay, as samples having <3log₁₀ GEC SMV were not detectable (Ct>45) by RT-qPCR. The captureefficiency of IMS/RT-qPCR (calculated as ((GEC of detected SMV byIMS-RT-qPCR)/(input GEC of SMV)×100)) was 1.7% at 4 log₁₀ GEC in 1 ml.

Discussion

Both the AMC-RT-qPCR method and the two-site binding sandwich-qPCRmethod had lower limits of detection approximating 1 log₁₀ (10) GEC SMV.For the AMC assay, the efficiency with which the aptamer-bound magneticbeads captured SMV improved with decreased virus titer, probably as afunction of saturation. Each assay design has its own advantages anddisadvantages. The two-site binding sandwich assay in particular haspotential utility as a screening tool. Specifically, this method (i) isnon-destructive of the target organism (virus); (ii) results in captureof the target for subsequent confirmatory analyses, if desired; and(iii) has the potential, with additional optimization, to be a moresensitive by virtue of the fact that more aptamers (which are aboutone-fourth the size of antibodies) can bind to each target virus. In ourassay design, we used qPCR amplification in place of theenzyme-substrate reaction of sandwich ELISA. As PCR produces exponentialamplification of the target, this may be a yet more sensitive means bywhich to amplify signal intensity. It should be noted that the antibodycapture step of the two-site binding sandwich method was the limitingaspect of the assay, as antibody-mediated capture efficiency was quitelow (<2%). This step also has disadvantages because HuNoV antibodiestend to have a high degree of specificity and hence lack broadreactivity. The capture efficiency and perhaps the assay sensitivity andreactivity could be improved if a more universal and efficient initialcapture step were used. Ligands such as porcine gastric mucin,histo-blood group antigens (HBGAs) and HBGA-like substances, and/orhuman plasma protein components can be useful alternatives to antibodiesin this assay design.

Example 3: Generation and Characterization of Nucleic Acid AptamersTargeting the Capsid P Domain of a Human Norovirus GII.4 Strain

Human noroviruses (NoV) are the most common cause of acute viralgastroenteritis worldwide (Glass et al., N. Engl. J. Med. 361:1776-1785(2009)) and the leading cause of foodborne illness in the United States(Scallan et al., Emerg. Infect. Dis. 17:7-15 (2011)). Despite theirpublic health significance, the availability of routine detectionmethods for these viruses is limited, in part due to the absence of anin vitro cultivation method. While molecular amplification (specificallyreverse transcriptase quantitative PCR or RT-qPCR) is usually used forNoV detection by the public health sector, it is not commonly used inclinical diagnostics. Perhaps because of sample complexity (fecalmatrix), ligand-based detection methods are more appealing for clinicaldiagnostics.

Unfortunately, human NoV are genetically and antigenically diverse,complicating the identification of broadly reactive ligands that can beused for virus capture and/or detection. For example, antibodies arewell documented to lack completely broad reactivity (Burton-MacLeod etal., J. Clin. Microbiol. 42:2587-2595 (2004); Shiota et al., J. Virol.81:12298-12306 (2007)), and for this reason, enzyme immunoassays displayrather poor sensitivity Costantini et al., J. Clin. Microbiol.48:2770-2778 (2010); Kele et al., Diagn. Microbiol. Infect. Dis.70:475-478 (2011). Other candidate NoV ligands have been explored, suchas putative NoV infection co-factors known as histo-blood group antigens(HBGAs; Cannon et al., Appl. Environ. Microbiol. 74:6818-6819 (2008);Harrington et al., J. Virol. 78:3035-3045 (2004)) and porcine gastricmucin, which contains some HBGAs (Pan et al., Food Microbiol. 30:420-426(2012); Tian et al., Appl. Environ. Microbiol. 74:4271-4276 (2008));peptides (Rogers et al., J. Clin. Microbiol. 51:1803-1808 (2013)); andsingle chain antibodies (Huang et al., Protein Eng. Des. Sel. 27:339-349(2014)). While some of these can react with multiple human NoV strainsor VLPs, none bind to all those tested.

For pathogen capture and purification, nucleic acid aptamers are apromising alternative ligand. Aptamers are short (20-80mer)single-stranded DNA or RNA sequences that interact (bind) to theirtarget through their three-dimensional structures. They offer advantagesover antibody-based affinity molecules in their ease of production,purification and modification, stability, and lower cost (Brody & Gold,Rev. Mol. Biotechnol. 74:5-13 (2000); Murphy, Nucleic Acids Res. 31:e110(2003); Tombelli et al., Biomol. Eng. 24:191-200 (2007)). Nucleic acidaptamers are selected in vitro based on affinity for a target molecule,protein, virus, or cell using a molecular-based iterative enrichmentmethod called SELEX (Systematic Evolution of Ligands by EXponentialenrichment).

In the absence of a robust in vitro cultivation method, the only sourceof whole viruses for ligand selection is stool samples from infectedindividuals. As infectious virus in stool is a difficult sample toobtain and work with, virus-like particles (VLPs) are frequently usedinstead for many types of studies, from disinfection to immune responsecharacterization (Cheetham et al., J. Virol. 81:3535-3544 (2007); Lou etal., Appl. Environ. Microbiol. 78:5320-5327 (2012); Nilsson et al.,Glycoconj. J. 26:1171-1180 (2009); Souza et al., J. Virol. 81:9183-9192(2007); Vongpunsawad et al., J. Virol. 87:4818-4825 (2013)). VLPsdemonstrate similar binding behavior to HBGAs as human NoV particles(Huang et al. 2003; White et al. 1996); however, their production andpurification can be costly, time consuming, and variable (Koho et al.,J. Virol. Methods 179:1-7 (2012)). An alternative is to focus selectionon a portion of the human NoV major capsid protein or VP1. Unlike VLPsfor which the entire capsid (all 180 copies of the major capsid protein(VP1)) assembles as “ghosts,” “P domain proteins” consist of a set ofproteins where the outermost domain of VP1 capsid proteins is expressed.Like VLPs and human NoVs, these proteins retain their antigenicity, canstill bind to histo-blood group antigens and have been used forstructural, binding, and vaccination studies (Cao et al., J. Virol.81:5949-5957 (2007); Koho et al., J. Virol. Methods 179:1-7 (2012); Tanet al., Procedia Vaccinol. 4:19-26 (2011)). P domain proteins can beproduced in a bacterial system (Tan and Jiang 2005) relatively easily,and expressed and purified at low cost and with high yield, making theman attractive target for ligand selection. In this study, we describethe production of single stranded (ss)DNA aptamers with binding affinityto a representative human NoV strain by SELEX using a P domain protein.Once isolated and characterized, promising aptamer candidates werefurther tested for their degree of reactivity with a broad panel ofhuman NoV VLPs. They were then used to develop prototype methods tocapture and/or detect GII.4 human NoV in outbreak-associated fecalspecimens.

Materials and Methods Viruses, Virus-Free Fecal Specimens, Virus-LikeParticles (VLPs), and Virus Capsid Protein

A GII.4 outbreak-derived human clinical (fecal) sample(sequence-confirmed to be the “2006b” cluster of GII.4 epidemic strains(Tsai et al., J. Med. Virol. 86:335-346 (2014); Yang et al., J. Virol.84:9595-9607 (2010)) was suspended 20% in phosphate-buffered saline(PBS). Human NoV-negative stool samples (confirmed negative by RT-qPCR)derived pre-exposure from individuals participating in a human challengestudy were also used. In some instances, stool suspensions were usedwithout further processing. In other cases, the suspensions werepartially purified by chloroform extraction (Shin & Sobsey, Water Res.42:4562-4568 (2008)). All suspensions were stored at −80° C. until usein experiments. The following virus-like particles (VLPs), whichconsisted of purified virus capsid without the viral genome, wereavailable for this study: GI.1, GI.4, GI.6, GI.7, GI.8, GII.1, GII.2,GII.3, GII.4 (2 strains), GII.6, GII.7, GII.12, and GII.17.

Preparation of P Proteins

Viral P proteins were selected as targets for SELEX. The genomic RNAfrom the GII.4 clinical outbreak stool specimen was extracted using thephenol-chloroform-ethanol precipitation method. Reverse transcriptionpolymerase chain reaction (RT-PCR) was performed using the commonlyused, broadly reactive JJV2F (Jothikumar et al., Appl. Environ.Microbiol. 71:1870-1875 (2005)) and G2SKR (Kojima et al., J. Virol.Methods 100:107-114 (2002)) primers. The two step RT-PCR was completedusing a DNA Engine (PTC-200) Peltier Thermal Cycler 200 (MJResearch/Bio-Rad Laboratories, Hercules, Calif.) with a 50° C. reversetranscription step for 30 min followed by enzyme inactivation bytreatment at 94° C. for 15 min. Amplification consisted of 35 cycles of94° C. for 30 sec, 58° C. for 50 sec, 72° C. for 60 sec, and a single72° final extension step for 5 min. Amplified cDNA was purified with theQIAquick PCR Purification Kit (Qiagen, Valencia, Calif.) and sequencedby Genewiz, Inc. (South Plainfield, N.J.). RT-PCR amplification andsequencing confirmed that the clinical outbreak stool specimen used forcreation of the P domain belonged to the 2006b GII.4 cluster. Primersspecific to the P domain region (nt 5744-6704), which included flankingBamHI and Nod restriction enzyme sites, were designed using the GII.42006b sequence (Accession Number: JN400603; Tsai et al., J. Med. Virol.86:335-346 (2014)) based on the locations of previously reported primerswithout a hinge (Tan & Jiang, J. Virol. 79:14017-14030 (2005)), and(Table 6). These were used to produce cDNA using the RETROscript kit(Ambion/Applied Biosystems) and amplified in PCR with the designedprimers (GII.4 P Domain Forward/Reverse, Table 6) and the Platinum Taqsystem (Invitrogen). Reactions were cycled at 95° C. for 90 sec,followed by 40 cycles of 95° C. for 30 sec, 55° C. for 40 sec, 68° C.for 90 sec, and a single final extension step of 68° C. for 5 min. Theproducts were then cleaned with the QIAquick PCR purification kit(Qiagen). After cleaning, 410 ng of the product was restriction digestedwith BamHI and NotI (New England BioLabs, Ipswtich, Mass.). This wasligated into a similarly digested pGEX-4T-1 plasmid (GE Healthcare,Piscataway, N.J.) containing an N-terminal glutathione-S-transferase(GST) tag with a 2:1 insert:vector ratio. The vector was thenelectroporated into electrocompetent E. coli BL21 (DE3) cells, whichwere plated on brain heart infusion (BHI) agar plates with 100 μg/mlampicillin and incubated at 37° C. for 24-48 h. Successful transformantswere screened by colony PCR and confirmed by sequencing (Genewiz, Inc.).

TABLE 6 Oligonucleotides Used in Selection and Characterizationof Aptamers with Binding Affinity to Noroviruses Name Sequence (5′-3′)SEQ ID NO DNA Aptamer AGTATACGTATTACCTGCAGC-(N)₄₀-CGATATCTCGGAGATCT 146Library TGC Sequence FAM-Forward FAM-AGTATACGTATTACCTGCAGC 147Constant Region Biotin-Reverse Biotin-GCAAGATCTCCGAGATATCG 148Constant Region Forward Constant AGTATACGTATTACCTGCAGC 147 RegionReverse Constant GCAAGATCTCCGAGATATCG 148 Region GII.4 P DomainGCACGGATCCTCAAGAACTAAACCATTTACTGTC 152 Forward* GII.4 P DomainGGACGCGGCCGCTTATAAAGCACGTCTACGCCC 153 Reverse* JJV2FCAAGAGTCAATGTTTAGGTGGATGAG 149 COG2R TCGACGCCATCTTCATTCACA 150Ring 2P Probe 56-FAM TGGGAGGGCGATCGCAATCT-3BHQ 151 T7GII.4FTAATACGACTCAACTATAGCAAGAGTCAATGTTTAGGTGGATGAG 154 GII.4R2GTTGGGAAATTCGGTGGGGACTG 155 *The underlined bases are restriction enzymerecognition sites.

P domain-GST fusion protein and GST-only cultures were grown overnightin 2× yeast extract tryptone ampicillin (YTA) broth incubated at 37° C.Thereafter, the bacteria were pelleted, reconstituted in 2×YTA, and usedto seed a larger 2×YTA culture that was grown at 37° C. to an OD₆₀₀ of0.6-0.9. The cultures were then induced with 1.0 mM isopropylβ-D-1-thiogalactopyranoside (IPTG) and left overnight at 25° C. withgentle shaking. Cells were purified by centrifugation and lysed by beadbeating. For further purification, the lysate was incubated 1:1 (v/v) in50% glutathione sepharose 4B agarose bead solution (GE Healthcare) for30-45 min at room temperature, followed by centrifugation and washing ofthe bead-protein complexes. Elution from the fusion protein was doneusing 50 mM Tris-HCl/10 mM reduced glutathione buffer (pH 8.0) mixed 1:1with the bead volume and incubated for 15-20 min at room temperaturefollowed by centrifugation. The presence of the P domain protein in thelysate and eluate was confirmed by Western blotting on nitrocellulosemembranes using anti-GST primary antibody (Thermo Fisher Scientific,Waltham, Mass.) and anti-GII.4 primary antibody (ab80024, Abcam,Cambridge, England).

Aptamer Selection (SELEX) and Characterization

Preparation of DNA Library.

An 81-base combinatorial DNA library having a 40 nt variable region wasprepared for SELEX by producing an 81 bp double-stranded (ds) DNAmolecule that was unlabeled at the 5′ end and labeled at 3′ end withbiotin by PCR using a Forward Constant Region primer and a biotinylatedReverse Constant Region primer (Table 6), as described previously byDwivedi et al., Appl. Microbiol. Biotechnol. 87:2323-2334 (2010). Forseparating the biotinylated DNA strand from its complementary strand,the labeled dsDNA was coupled with Streptavidin MAGNESPHERE paramagneticparticles (Promega), and captured by magnet (MPC-M magnetic particleconcentrator, Dynal A.S. Oslo, Norway). The captured dsDNA was denaturedby treatment with 0.15 M sodium hydroxide and after three washes withTris-EDTA, the immobilized biotinylated strand was released byincubating beads in 28% ammonium hydroxide at 85° C. for 10 min. Removalof residual ammonium hydroxide was achieved using Vivaspin 500 filters(10,000 molecular weight cut-off, Sartorius Stedim Biotech, Cedex,France) with two washes of nuclease-free water. The purified ssDNA wasstored in −80° C. until use.

Selection of Aptamers Using GII.4 HuNoV P Protein.

SELEX and counter SELEX were done using the P domain-GST fusion proteinand the GST tag with NoV-negative human stool and bead matrix astargets, respectively. Briefly, 300-500 pmol of library was pre-heatedat 90° C. for 10 min and cooled on ice for 10 min. For counter SELEX,the library was exposed to a 125 μl bed volume of the GST beads for 1 hat room temperature with end-over-end mixing. The mixture wascentrifuged at 500×g for 5 min and the supernatant reserved. DNA waspurified by phenol:chloroform:isoamyl alcohol (25:24:1) extraction andethanol precipitation (10% (v/v) 3 M sodium acetate, 200% (v/v) 100%ethanol, and 50 μg/ml Ambion GlycoBlue (Life Technologies, Grand Island,N.Y.)) with reconstitution of the pellet in 25 μl DEPC-treated water.The DNA concentration was adjusted to 20-40 ng/μ1 and amplified by PCRusing 2 μl of the template and the PCR primers described in Table 6. Thereactions of 50 μl contained 1×GOTAQ Buffer (Promega), 500 nM ofConserved Forward Primer, 500 nM biotinylated Conserved Reverse Primer,0.2 mM PromegaPCR Nucleotide mix (Promega), 0.5 μs single-stranded DNAbinding protein (Promega), and 2 U GOTAQ DNA polymerase (Promega). ABio-Rad T100 Thermal Cycler was used for the PCR (Bio-Rad Laboratories,Hercules, Calif.) with an initial 95° C. step for 2 min; 30 cycles of95° C. for 30 s, 50-65° C. for 30 s (see below), and 72° C. for 15 s;and a final extension at 72° C. for 5 min. After each round of SELEX andcounter-SELEX, an initial annealing gradient (from 50-65° C.) using thecycling conditions above was used to determine the optimal annealingtemperature prior to the larger regeneration of the remaining pool. Thistemperature optimization was required to reduce concatamers and primerdimers. The amplified pool was then made into biotin-labeled ssDNA asdescribed above.

The initial counter-SELEX was followed by seven rounds of positiveselection, which were performed in the same manner as the counter-SELEXdescribed above except that the P domain-GST fusion protein lysate wasused instead of the GST lysate; unbound sequences were removed bywashing; and the protein-aptamer complexes were eluted from the beadsusing a glutathione elution buffer (50 mM Tris-HCl/10 mM reducedglutathione buffer (pH 8.0)) followed by phenol-chloroform extractionand ethanol precipitation. Prior to sequencing, another counter-SELEXround was performed using GST lysate and human NoV-negative human stool.The amplified pool was then resolved on a 2% agarose gel and purifiedwith the QIAquick Gel Extraction Kit (Qiagen). The purified pool wascloned via electroporation using the TOPO TA Cloning Kit (Invitrogen).Colonies were selected, grown, plasmid-extracted, and screened by PCR.Selected colony plasmids were then sequenced (Genewiz, Inc.).

Analysis of Aptamer Sequences, Structural Folding, and Stability.

Usable aptamer sequences obtained were grouped into identical/similarsequences, and the proportion of each sequence in the pool determined.Structural folding analysis and ΔG prediction of the candidate aptamersequences was performed using the DNA Mfold online server using 0.5 mMmagnesium, 1 mM sodium, and 23° C. as input parameters (Zucker, NucleicAcids Res. 31:3406-3415 (2003)). Candidate sequences from the pool wereselected on the basis of how many times they repeated in the pool, lowΔG value (stability), and uniqueness and formation of loops in thesecondary structure. Common sequence identification of the randomregions of the aptamers in the sequence pool was conducted using theMEME server for analysis of only the input aptamer strands with aminimum motif length of 6 bases (Bailey & Elkan, Proc. Int. Conf.Intell. Syst. Mol. Biol. 2:28-36 (1994); Bailey et al., Nucleic AcidsRes. 37:W202-W208 (2009)). Motif analysis was conducted for fourrepresentative random regions (M1, M5, M6-2, and M9-2; see Table 7), toexclude some random regions that contained slight base substitutionsand/or additions that would have generated redundant memes.Additionally, a MEME analysis was run comparing only the two sequencerandom regions chosen for further characterization (M1 and M6-2). Motifsidentified by MEME analysis were considered as potential motifs if theycontained no more than two mismatches within at least a set of six basesbetween two or more sequences.

Evaluation of Aptamer Binding Using an Enzyme-Linked Aptamer SorbentAssay (ELASA)

Binding affinity assays were done using the candidate aptamers and apanel of virus-like particles (VLPs) corresponding to genogroup I (GI.1(Norwalk virus), GI.2, GI.4, GI.6, GI.7 and GI.8) and genogroup II(GII.1, GII.2 (SMV), GII.3, GII.4 (Houston and Grimsby), GII.6, GII.7,GII.12 and GII.17) HuNoV, and also for chloroform-extracted 20% stoolsuspensions derived from a patient confirmed to have GII.4 New Orleansinfection. This was done using a previously reported ELISA-like method(Escudero-Abarca et al., PLoS One 9:e106805 (2014); Moe et al., Clin.Diagn. Lab. Immunol. 11:1028-1034 (2004); Rogers et al., J. Clin.Microbiol. 51:1803-1808 (2013)). We refer to this assay as Enzyme-LinkedAptamer Sorbent Assay (ELASA). Briefly, VLP suspensions (1.3-4.3 mg/ml)were adjusted to a concentration of 3 μg/ml in PBS; in the case of wholevirus, 10-fold serial dilutions of chloroform-extracted 20% GII.4 NewOrleans stool solutions were made. One hundred μl aliquots of VLP ordiluted stool were placed on the bottom of a flat-bottom, polystyrene96-well plates (Costar 3591, Fisher, Pittsburgh, Pa.) along with 100 μlPBS in other no-VLP-control wells, and plates were incubated overnightat 4° C. After removal of the fluid, the wells were blocked with 200 μlof 5% skim milk in PBS-Tween 20 (0.05%) (PBST) with a 10 nM mix ofunrelated DNA oligonucleotides (Listeria monocytogenes primers hlyQF/Rand L23 SQF/R (Rodriguez-Lázaro et al., Appl. Environ. Microbiol.70:1366-1377 (2004)) for 2 h at room temperature with gentle shaking.Blocking solution was discarded, and three washes of 200 μl PBST perwell were performed. Next, 100 μl of biotinylated aptamer (1 μM) wasadded to each well, and the plate was incubated for 1 h at roomtemperature with gentle shaking. After removal of the liquid, the plateswere washed 4 times with PBST. One hundred μl of ELISA-gradestreptavidin-horseradish peroxidase (1 mg/ml, 1:5000, Invitrogen,Carlsbad, Calif.) was added per well with incubation for 15 min at roomtemperature with shaking. After removing the unbound enzyme andrewashing with PBST, 100 μl of 3,3′,5,5′-Tetramethylbenzidine (TMB)microwell peroxidase substrate system (solution A:B (1:1), KPL,Gaithersburg, Md.) was added for color development, and absorbance at450 nm was recorded using a microplate reader (Tecan Infinite M200pro,Tecan Group Ltd., Mannedorf, Switzerland).

Plate Data Analysis

All ELASAs were replicated on three separate occasions with at leastthree wells per replicate. Results were expressed as the ratio betweenthe absorbance values for test samples divided by those for the negativecontrol (no VLP). As per convention (Ebel et al., Emerg. Infect. Dis.8:979-982 (2002); Escudero-Abarca et al., PLoS One 9:e106805 (2014);Hirneisen & Kniel, J. Virol. Methods 186:14-20 (2012)), a VLP/no VLPratio of less than 2.0 was considered to be low-to-no binding (−); aratio from 2.0 to 5.0 was considered to be low binding (+/−); 5.0 to10.0 was considered to be medium binding (+); and ≥10.0 to be strongbinding (++). Means and standard deviations for ratios associated withreplicate experiments were calculated using Microsoft Excel. For theplates containing positive and negative chloroform-extracted stool,statistical comparison was performed using a one-way analysis ofvariance (ANOVA) with Tukey's multiple comparison using GraphPad Prismversion 5.0d.

Aptamer Magnetic Capture (AMC) and RT-qPCR

Biotinylated aptamers were used to concentrate human NoV from stoolsamples. Thirty μg of DYNABEADS MYONE Streptavidin C1 magnetic beads(Invitrogen-Dynal AS, Oslo, Norway) were diluted in 1 ml PBS+0.05% PBST,mixed, and recaptured using the Dynal MPC-M magnetic particleconcentrator (Invitrogen-Dynal). The beads were resuspended in 1 ml of5% skim milk and blocked overnight at 4° C. with rotation. The beadswere then washed with 500 μl PBST twice, resuspended in 50 μl PBST, andstored at 4° C. until use. These beads will hereafter be referred to as“blocked beads.”

Aptamer Magnetic Capture of Human Norovirus from Stool Specimens.

Aptamer capture of human NoV from stool was performed based on theprotcol of Cannon et al., Appl. Environ. Microbiol. 74:6818-6819 (2008)with substitute of aptamers for purified histo-blood group antigens(HBGAs). Ten-fold serial dilutions of a previously aliquoted 20% GII.4stool suspension were prepared in PBS, and 100 μl of each dilution wasplaced into a dedicated tube containing 900 μl PBST and 15 μl ofbiotinylated aptamer (100 μM, ˜5.9 ng total). The contents were mixed byend-over-end rotation for 1 h at room temperature. Fifty μl of theblocked beads were then added, and the tubes incubated for another hourwith flipping at room temperature. Beads were magnetically recovered andwashed once with 500 μl PBST followed by one wash with 500 μl PBS. Beadswere resuspended in 100 μl PBS and stored at −80° C. until RNAextraction. Negative controls consisted of tubes containing 450 μl PBST,450 μl Superblock T20 (Thermo Fisher Scientific, Waltham, Mass.), 100 μlof diluted sample, and 500 of blocked beads. RNA extraction was doneusing the NUCLISENS EASYMAG system (bioMerieux SA, Marcy l'Etoile,France) according to the manufacturer's instructions with a 40 μl finalelution volume. The eluted RNA was immediately stored at −80° C. untiluse in RT-qPCR (below).

Quantification of Viral Recovery by RT-qPCR

RNA was amplified by one step RT-qPCR using the SUPERSCRIPT III PLATINUMOne-Step kit (Invitrogen). Reactions of 25 μl were made containing 12.5μl 2× Reaction Mix, 0.5 μl SUPERSCRIPT III ReverseTranscriptase/PLATINUM Taq mix, 200 nM JJV2F primer, 200 nM COG2Rprimer, 200 nM Ring2P probe (Jothikumar et al., Appl. Environ.Microbiol. 71:1870-1875 (2005)), 5.5 μl nuclease-free water, and 50 μltemplate. Reverse transcription was done at 50° C. for 15 minutes,followed by enzyme inactivation at 95° C. for 2 minutes. Amplificationwas done for 45 cycles of 95° C. for 15 seconds, 54° C. for 30 seconds;and 72° C. for 30 seconds. Quantification of genomic copies was basedupon a standard curve using an in vitro transcribed GII.4 New Orleansamplicon covering a 460 nt range of the genome containing theJJV2F-COG2R primer target region. The amplicon was quantified using ananodrop, serially diluted, and used to construct a standard curve toestimate genomic copies. Amplifiable RT-qPCR units were estimated basedon a standard curve of Ct values from serial 100 μl dilutions of the 20%GII.4 New Orleans stool isolate used for the AMC assay that had theirgenome RNA extracted and amplified using RT-qPCR as described above.

Aptamer Candidate Selection

After seven rounds of SELEX and two rounds of counter-SELEX, aptamerpools were sequenced, resulting in a pool of 11 sequences and leading tothe identification of six unique aptamer candidates. All are detailed inTable 7. Candidates M1 and M6-2 were selected for furthercharacterization based on the number of times the sequence occurred inthe pool of sequences (3/11 and 1/11, respectively), low dG values(dG=−7.11 Kcal/mol and dG=−8.33 Kcal/mol for M1 and M6-2, respectively),and similarities in secondary structure. Candidates M1 and M6-2 havesecondary structures containing multiple loops; however, both have someloops that contain conserved library primer regions involved information of stem-loops (FIGS. 12 and 13, respectively). FIG. 14 showsthe predicted structural folding of aptamer M5 (dG=−6.43 Kcal/mol) withconstant regions attached.

TABLE 7 Representative Aptamer Sequences Obtained from 7^(th) Round ofSELEX Against GII.4 P Domain and Two Rounds of Counter-SELEXAgainst GST-Beads and Negative Human Stool Round of SEQ # Aptamer SELEXdG Random Region Sequence^(a) ID NO Repeats Identifier 7 −7.11TGTTTATGGGGATAAACGTATCTAATTCGTGTACTAATCA 35 3/11 M1^(ab) 7 −4.12TGTTAAGGGGAATTAATAATGATAATCCGTCTACTAATCA 36 2/11 M9-2 7 −3.95TGTTAGGGGGAATTAATAATGGATAATCCGTCTACTAATC 37 1/11 M12-1 A 7 −8.13TGGGGGGTGGTGCGGTGTGTGGCAGGGGAGCATAGCCGGG 38 1/11 M13-2 GGCCCCCT 7 −8.33TGGGAAGAGGTCCGGTAAATGCAGGGTCAGCCCGGAGAG 39 1/11 M6-2^(ab) 7 −6.43TGGGGGGTGGTGCGGTGTGTGACAAGGGAGCATAGCCGG 40 3/11 M5^(b) GGGCCCCCT^(a)Candidate aptamers selected for further characterization ^(b)Chosenfor MEME analysis using MEME 4.9.1

MEME analysis showed multiple overlapping motifs involved in loopregions or the formation of loop regions. Sequence motif analysis ofthree aptamer random regions from the pool (which excluded randomregions that were nearly identical to another region with the exceptionof one or two base substitutions/additions) as well as the two selectedaptamers (M1 and M6-2) yielded several potential motifs (motifs 4-9) ofat least 6 bases shared between different pairs of aptamers, allowingtwo mismatches. Other motifs were identified through further analysis.Motif 4 was shared between aptamers M1 and M6-2 and contained thesequence TGGGNA (SEQ ID NO: 101). The motif occurs near the beginning ofthe random regions of both of the aptamers and is involved in majorloops in both of the aptamers. This is potentially a binding/interactivesite, as stem-loop structures are targeted as potential binding sites onaptamers when screening to create truncated aptamers (Kaur et al., PLoSOne 7:e31196 (2012)). Interestingly, this same motif region is alsoshared with M5, though the arbitrary cut-off of no more than two basemismatches in a motif means it could not be combined to be shared by allthree aptamers (the motif shared by all three aptamers would be TGGGRRKusing IUPAC nomenclature, which defines R as A or G and K as G or T)(motif 13; SEQ ID NO: 110). Motif 6 (TGGGNNG; SEQ ID NO: 103) is sharedbetween M5 and M6-2, and occurs in a similar position on the twoaptamers. Motif 6 is involved in loop regions on both aptamers, likemotif 4. This suggests that the two similar motifs (4 and 6; TGGGRRK)comprise a sequence important for binding to HuNoV. Motif 5 (GNGANAA,SEQ ID NO: 102) is shared between M1 and M5, and occurs in the same loopregion affected by the aforementioned conserved TGGGRRK loop sequencefor M1. Motif 7 (TCNNGTA; SEQ ID NO: 104) is shared between M1 and M6-2.It occurs in the same loop as the conserved TGGGRRK sequence in M6-2,but appears as part of a separate stem-loop region in M1. Motif 7 occursnear the beginning of the random regions of both aptamers and isinvolved in their major loops. Motif 8 (GGTNCGGT; SEQ ID NO: 105) isshared between M5 and M6-2 at the same location and is involved in themajor predicted stem-loops of both structures. It is also located nearthe conserved TGGGRRK sequence of both aptamers, but the resulting loopsappear notably different based in secondary structure position in Mfold.Motif 9 (TAAANGNA; SEQ ID NO: 106) occupies similar positions on both M1and M6-2. It is involved in forming the end of the stem and beginning ofthe conserved TGGGRRK loops of both of the aptamers. Motif 9 is involvedin the stem-loop regions for the major loop of both aptamers.

It is interesting to note that many of the observed motifs occur in theloop regions of the aptamers, and many different motifs overlap. Thismight imply that different common sequence elements were conserved inthe SELEX pool, but occur at different positions on the three differentaptamers. M6-2 contains many overlapping motifs, all involved in a majorstem loop, suggesting that the sequence was selected because itcontained an “amalgamation” of different selected binding elements seenon the other two aptamers. The overlapping motifs are possibly theresult of these conserved yet slightly different elements seen indifferent locations of the aptamers. If less stringent base mismatcheswere allowed, then a large sequence could be created that is sharedbetween the three aptamers. For example, if the same base in two out ofthe three aptamers at each base position were the minimum, then thefollowing sequence would be present in aptamers M1, M5, M6-2, and M13-2:TGGGRRKWRRYSYRKY (R=A or G; K=G or T; W=A or T; Y=C or T; S=G or C)(motif 14; SEQ ID NO: 111). If restricted to just the conserved motifamong M5, M6-2, and M13-2, a motif could be constructed that would havethe sequence TGGGRRGWGGTSCGGT (SEQ ID NO: 174).

Notable sequence similarity between M1, M9-2, and M12-2 was detected.M1, M9-2, and M12-2 have very similar sequences in bases 1-16 and 23-40of their random regions, indicating that these regions may be importantfor aptamer binding. These sequences have been designated motifs 10 and11, respectively. Motif 10 has the sequence TGTTNNNGGGNATNAA (SEQ ID NO:107), and motif 11 has the sequence TAATNCGTNTACTAATCA (SEQ ID NO: 108).

Aptamer Binding Inclusivity

Both aptamer candidates (M1 and M6-2) exhibited relatively strongerbinding to VLPs representing GII human NoV genotypes over GI genotypes(Table 8). In Table 8, the values indicate the ratio between absorbancereadings for the test sample versus negative control (VLP wellsabsorbance/no VLP wells absorbance) for each aptamer. Results less than2.0 are considered to be negative (−) per convention for determiningassay detection limits and hence binding affinity; results in between2.0-5.0 are considered low binding (+/−); results in between 5.0 and10.0 are considered medium binding (+), and results >10.0 are consideredstrong binding (++). Values obtained for the negative control were inthe range of 0.1-0.4. All experiments were done with at least threewells per replicate in triplicate. For aptamer M1, strong binding (++)was observed for GII.2 and GII.4 (Houston) while medium binding (+) wasobserved for GI.7, GII.4 (Grimsby), and GII.7 VLPs. For aptamer M6-2,strong binding (++) was seen for GII.2 and both GII.4 VLPs, and GI.7,GII.7, GII.12, and GII.17 VLPs bound to the aptamer with mediumintensity. Based on an absorbance ratio cutoff of 2.0, aptamer M6-2exhibited broader reactivity compared to aptamer M1, with some degree ofbinding to all of the VLPs tested. Positive signals were quite low forGI.6 and GII.3 VLPs. On the other hand, aptamer M1 did not appear tobind to GI.6, and had relatively low signals for GI.8, GII.3, and GII.6VLPs. Overall, M6-2 also had higher VLP/No VLP ratios compared to M1. Asexpected, assays using GII.4 VLPs provided some of the highest signalratios. Interestingly, both aptamers also had GII.2 ratios about as highas the GII.4 (highest) VLPs.

TABLE 8 Binding Affinity of Selected Aptamers Against a Broad Panel ofNorovirus VLPs VLPs Average VLP Pos/No VLP Ratio (Std. Dev.)^(a)Aptamers Genogroup M1 M6-2 GI.1 (Norwalk)  3.40 (0.67) (+/−)  4.28(0.52) (+/−) GI.4  3.17 (0.43) (+/−)  3.36 (0.24) (+/−) GI.6  1.98(0.38) (−)  2.75 (0.45) (+/−) GI.7  7.32 (2.41) (+)  7.56 (2.45) (+)GI.8  2.61 (0.26) (+/−)  4.55 (0.81) (+/−) GII.1  3.59 (1.55) (+/−) 4.52 (0.44) (+/−) GII.2 (Snow Mountain) 10.68 (0.70) (++) 12.00 (1.10)(++) GII.3  2.94 (1.90) (+/−)  3.16 (0.72) (+/−) GII.4 (Grimsby)  7.59(0.46) (+) 11.54 (1.70) (++) GII.4 (Houston) 10.41 (1.23) (++) 12.98(1.76) (++) GII.6  2.38 (0.78) (+/−)  4.40 (0.85) (+/−) GII.7  7.47(1.15) (+)  8.02 (1.99) (+) GII.12  4.47 (0.54) (+/−)  5.55 (0.10) (+)GII.17  3.94 (1.03) (+/−)  5.24 (0.75) (+) ^(a)Values indicate the ratiobetween absorbance readings for test VLP sample versus negative control(VLP wells absorbance/No VLP wells absorbance) for each aptamer.

Different binding patterns for these two aptamers indicate that they maybind to different regions of the P protein and hence the viral capsid.For example, it is possible that aptamer M1 binds the P2 subregion ofthe P domain because it is not as broadly reactive as aptamer M6-2. TheP2 subregion can be involved in host cell binding and can behypervariable, responsible for antigenic drift of GII.4 strains(Lindesmith et al., PLoS Med 5:e31 (2008)). Further, aptamer M6-2 showedhigher signal intensity for all VLPs to which the two aptamers bound,and much higher intensity for the two GII.4 VLPs (Houston and Grimsby).Both aptamers bound well to the more recent GII.4 Houston strain, butaptamer M1 appears to have a noticeably lower affinity to the GII.4Grimsby strain relative to M6-2. The target of SELEX was a GII.4 strainfrom 2007, thus higher binding to the Houston strain may be expected asHouston (2001) has less evolutionary distance from the 2006b target thanGrimsby (1996; (Glass et al., N. Engl. J. Med. 361:1776-1785 (2009);Shanker et al., J. Virol. 85:8635-8645 (2011).

Interestingly, both M1 and M6-2 aptamers bound GII.2 Snow Mountain VLPsabout as well as the GII.4 strains. It may be possible that the aptamersare binding a conserved region between GII.4 and GII.2 Snow Mountainvirus (SMV). This is further supported by the fact that aptamersproduced against SMV also exhibited strong binding to Houston andGrimsby VLPs (see Example 1). However, it is also possible that thesimilar binding may be related to the preparation or characteristics ofthe VLPs, as the same VLP panel was used in both studies.

Aptamers Bind to Partially Purified Human Stool Samples Obtained fromInfected Individuals

Both the M1 and M6-2 aptamers exhibited binding to serially dilutedpartially purified 20% stool specimens obtained from infectedindividuals (FIG. 15). Binding was statistically significant (p<0.05)relative to human NoV negative stool when the samples were diluted 10⁻²or 10⁻³. These differences were not statistically significant for the10⁻¹ dilutions of stool, likely due to matrix-associated non-specificbinding. When stool samples were diluted 10⁻⁴ or more, signal was lost,presumably because of dilution-associated depletion of virus,approaching the assay limit of detection.

Aptamer Magnetic Capture (AMC) Coupled to RT-qPCR Applied to OutbreakStool Specimens

Aptamers M1 or M6-2 were used to concentrate HuNoV from diluted GII.4New Orleans clinical stool isolates using magnetic nanoparticles.Concentrated viruses were then quantified by RT-qPCR (FIG. 16). Negativecontrols consisted of beads without the aptamer conjugates. An asteriskdesignates statistical significance (p<0.05) when comparing aptamer andno aptamer controls. Both aptamers concentrated significantly more virus(p<0.05) than no aptamer controls at concentrations of 5.86 and 6.79log₁₀ human NoV genomic units per ml of stool. At virus concentrationslower than this, the aptamers failed to produce statisticallysignificant differences compared to the controls, although this could bedue to increased variability at lower virus concentrations.

Use of the cloned P domain as a target for SELEX has potential benefitfor the development of future diagnostics. With HuNoV capsid proteinunder constant selective pressure, a cost-effective, rapid, and easilyimplemented method for the creation of large quantities of bindingligands with high affinity to emerging strains could speed thedevelopment of detection assays. The generally low cost and instrumentalrequirements of cloning and expressing the P domain in the E. coli modelproduced an abundant source of target without the need for specializedequipment, or access to human stool from infected individuals; all thatwould be needed is capsid sequence information. Furthermore,demonstration of rapid, microfluidic SELEX processes may furtherfacilitate the rapid identification of ligands specific to proteintargets, which might include the P domain of HuNoV (Huang et al.,Biosens. Bioelectron. 25:1761-1766 (2010); Lou et al., Proc. Nat'l Acad.Sci. U.S.A. 106:2989-2994 (2009)).

Discussion

In this example, the P domain cloned and expressed from the genome of a2007 GII.4 human NoV clinical stool isolate was used as the target inSELEX for production of ssDNA aptamers. A GII.4 strain was consideredrelevant because it belongs to the epidemic genotype that has beencausing the largest numbers of outbreaks and cases over the last twodecades (Bok et al., J. Virol. 83:11890-11901 (2009); Noel et al., J.Infect. Dis., 179:1334-1344 (1999)). Furthermore, the P2 subdomain ofthe P domain is thought to be involved in host cell binding, ishypervariable, and likely responsible for antigenic drift of GII.4strains (Lindesmith et al., PLoS Med 5:e31 (2008)). Given thesefeatures, the aptamers produced in this study did not strongly bind toall human NoV VLPs screened. However, they did exhibit binding to amajority of the VLPs tested, with generally better binding demonstratedfor GII versus GI VLPs. A notable difference between aptamers M1 andM6-2 was observed for GI.8, GII.6, and GII.17 VLPs (Table 8). Differentbinding patterns for these two aptamers suggest that they may bind todifferent regions of the P domain. It could be hypothesized that aptamerM1 binds a less conserved region of the P domain because it was not asbroadly reactive as M6-2, and overall showed lower signal intensity. Asexpected, both aptamers bound well to the GII.4 VLPs, as the aptamertarget was a GII.4 2006b strain. Also as expected, both aptamersdisplayed stronger binding to the more recent and sequentially similarGII.4 Houston strain (2001) compared to the older and less sequentiallysimilar GII.4 Grimsby (1996) strain (Glass et al., N. Engl. J. Med.361:1776-1785 (2009); Shanker et al., J. Virol. 85:8635-8645 (2011)).Interestingly, M6-2 showed some degree of binding to all of the VLPstested, suggesting that it likely binds a part of the P1 subdomain ofNoV, where other fairly broadly reactive antibodies have been found tobind (Kitamoto et al., J. Clin. Microbiol. 40:2459-2465 (2002); Parkeret al., J. Virol., 79:7402-7409 (2005); Shiota et al., J. Virol.81:12298-12306 (2007)).

Multiple common sequence motifs within the variable region of aptamersM1 and M6-2 were identified. Many of these motifs are involved instem-loop or loop structures (FIGS. 12 and 13, respectively) and may beimplicated in aptamer binding to human NoV, as loop and stem-loopstructures are often involved in binding (Kato et al., Nucleic AcidsRes. 28:1963-1968 (2000); Kaur et al., PLoS One 7:e31196 (2012)). Suchmotif analysis can inform future studies. For example, characterizationof the nature of the aptamer binding domain(s) could be furtherinvestigated using nucleotide substitution. Further, identification ofcommon motifs in aptamers M1 and M6-2, in addition to other aptamersmight allow the production of truncated aptamers that could be combinedinto a chimeric aptamer (Kanwar et al., Crit. Rev. Biochem. Mol. Biol.46:459-477 (2011)) to create an even more effective broadly reactiveligand.

Recently, three studies have reported the development of DNA aptamershaving binding affinity to NoV. Giamberardino et al., PLoS One 8:E79087(2013) produced aptamers targeting the murine norovirus (MNV) surrogateusing whole virus SELEX, finding that one also bound GII.3 NoV VLPs;this aptamer was used as a recognition element in a voltammetry-basedbiosensor. Beier et al., FEMS Microbiol. Lett. 351:162-169 (2014)created DNA aptamers using an unspecified GII.4 strain's entire majorcapsid protein (VP1) by a different SELEX process than ours. The reportof that work focused primarily on innovations in bioinformatic analysisand protein-aptamer modeling rather than the functional bindingcharacterization reported here. Interestingly, the aptamers produced byGiamberardino et al. (2013) and Beier et al. (2014) had AG valuessimilar to M1 and M6-2, but the sequences and secondary structuresdiffered from ours. In both papers, the aptamers produced were neverapplied for capture or detection of human NoV in outbreak-derived stoolspecimens, and aptamer binding to the intact capsid of only one genotypeof human NoV was confirmed for any of the reported aptamers.

In another study (see Example 1 and Escudero-Abarca et al., PLoS One9:e106805 (2014) created aptamers using partially purified infectiousGII.2 Snow Mountain virus from stool (whole virus SELEX), as juxtaposedto the GII.4 P domain target in this example. The aptamers described inthat study, as well as those reported in this example, exhibited similarbroad reactivity and high signal-to-noise ratios, despite thedifferences in target. The aptamers also had similar AG values andmotifs that also occurred in stem-loops. Likewise, similar bindingsignals were observed using partially-purified GII.4 stool isolate inthe ELASA assay. Aptamers M1 and M6-2 exhibited lower captureefficiencies in AMC-RT-qPCR compared to aptamer 25 reported byEscudero-Abarca et al. (2014) (Example 1). This may be a function ofdifferences in the counter-selection process, as Escudero-Abarca et al(2014) (see Example 1) performed more counter-SELEX rounds against agreater number of targets, which likely reduced nonspecific aptamerbinding to magnetic particles and stool components. Nonetheless, theaptamers M1 and M6-2 displayed a reasonably good capture efficiency at arange of 4.88-6.79 log₁₀ input genomic copies of virus. Because of thesimilar performance of the M1, M6-2, and the Escudero-Abarca et al.(2014) aptamers (see Example 1) by ELASA and AMC-RT-qPCR, it is possiblethat they all bind to a conserved region, but further analysis would benecessary to support this hypothesis. The limit of detection of theAMC-RT-qPCR assay was 4.88 log₁₀ input genomic copies, whichcorresponded to about 2-3 log₁₀ RT-qPCR amplifiable units in the inputstool sample.

Unlike any of the previous reports of aptamer generation to NoV, thispaper is the first report of aptamers developed with a biotin label.Label modifications made after the development of aptamers have thepotential to reduce aptamer binding affinity (Jiang et al., Anal. Chem.76:5230-5235 (2004); Wang et al., Anal. Chem. 77:3542-3546 (2005)); thusselection using a functional biotin label allows for many downstreamdiagnostic and detection applications with less risk of losing aptamerfunctionality.

Not only were aptamers M1 and M6-2 able to bind to multiple human NoVVLPs, they also bound to stool samples previously confirmed as positivefor human NoV as evaluated by both the ELASA and AMC assay. The stoolspecimens were purified and diluted in order to achieve reliabledetection signals; matrix-associated interference with ligand bindingmay occur when samples are too “dirty.” This may be due to a degree ofnon-specific binding and/or association of the aptamers with theextracted stool matrix. This phenomenon has been observed in similartypes of assays done by other investigators for both aptamers(Escudero-Abarca et al., PLoS One 9:e106805 (2014); see Example 1) andother ligands (Burton-MacLeod et al., J. Clin. Microbiol. 42:2587-2595(2004); Huang et al., Protein Eng. Des. Sel. 27:339-349 (2014); Li etal., J. Food. Prot. 75:1350-1354 (2012)). Interestingly, the dilution ofthe chloroform-extracted stool to about 0.2% original stool content forGII.4 New Orleans is similar to the optimal 1% stool dilution reportedby Huang et al. (2014) when detecting NoV GII.4 in ELISA using phagesdisplaying single-chain antibodies. When it comes to AMC, non-specificbinding to the paramagnetic beads is commonly observed, as has beenreported by others for bacteria (Rijpens et al., Int. J. Food Microbiol.46:37-44 (1999); Tomoyasu, Appl. Environ. Microbiol. 64:376-382 (1998))and NoV (Escudero-Abarca et al., PLoS One 9:e106805 (2014); GI.Ipatricket al., J. Virol. Methods 90:69-78 (2000)). All told, regardless of theligand or assay design, non-specific binding often impacts analyticalsensitivity and this remains a recalcitrant issue for development ofrapid, reliable, and sensitive human NoV detection methods.

The human NoV capsid protein is under constant selective pressure,especially GII.4 strains, and strain emergence occurs every few years(Bull et al., PLoS Pathog. 6:e1000831 (2010); Debbink et al., J. Virol.86:1214-1226 (2012)). With respect to development of advanced detectionand vaccination strategies that can cover emerging strains, it isimportant to have a readily available target for product developmentpurposes. A functional human NoV P domain can be easily cloned,expressed and purified in E. coli with only capsid sequence informationneeded, thus resulting in the production of high concentrations ofprotein at low cost with relative ease. In short, the method describedhere can provide a cost-effective, rapid, and easily implemented meansto create large quantities of ligands with high affinity to emerginghuman NoV strains. As rapid, microfluidic SELEX processes emerge, thismay become an even simpler and faster means by which to select ligandswith binding specificity to protein targets (Huang et al., Biosens.Bioelectron. 25:1761-1766 (2010); Lou et al., Proc. Natl. Acad. Sci.U.S.A. 106:2989-2994 (2009)).

In summary, we isolated and characterized ssDNA aptamers with bindingspecificity to a broad range of human NoV VLPs and outbreak strainsusing an E. coli-expressed viral capsid protein, and demonstrated thatthey could be used as capture ligands in both ELISA-type andaptamer-mediated magnetic capture-RT-qPCR assays. The aptamers reportedhere are among the broadest reacting ligands to human NoV identified todate (Escudero-Abarca et al., PLoS One 9:e106805 (2014) (see Example 1);Hardy et al., Virology 217:252-261 (1996); Huang et al., Protein Eng.Des. Sel. 27:339-349 (2014); Kitamoto et al., J. Clin. Microbiol.40:2459-2465 (2002); Kou et al., Clin. Vaccine Immunol. 3849 (2014); Liet al., Virus Res. 151:142-147 (2010); Shiota et al., J. Virol.81:12298-12306 (2007); Yoda et al., J. Clin. Microbiol. 41:2367-2371(2003); Yoda et al., BMC Microbiol. 1:24 (2001)). With furtherdevelopment, the aptamers may be useful in novel detection platformssuch as biosensors (reviewed in Torres-Chavolla & Alocilja, Biosens.Bioelectron. 24:3175-3182 (2009)) and also may have relevance inantiviral or therapeutic applications (Jeon et al., J. Biol. Chem.279:48410-48419 (2004); Khati et al., J. Virol. 77:12692-12698 (2003);Yoon et al., Antiviral Res. 88:19-24 (2010)). This is the first reportto demonstrate that broadly reactive aptamers binding human NoV can beeasily and cheaply produced using SELEX directed against the P domain ofthese viruses.

Example 4: Selection and Characterization of Aptamers with BindingAffinity to the Norwalk Virus P Protein Materials and MethodsPreparation of Targets for Aptamer Selection

Virus Suspensions.

Norwalk virus (NV) was obtained as fecal extracts suspended 20% inphosphate-buffered saline (PBS) originating from human challengestudies. NV was used without further purifications.

Construction, Expression, and Purification of P Particles.

Norwalk virus P domain (corresponding to capsid amino acid residues226-530) was amplified using primers P493 and P494 (Table 9) (Tan etal., J. Virol. 78:6233-6242 (2004)). The resulting PCR product wascloned as a BamH1-NotI fragment into vector pGEX-4T-3 (GE Healthcare,Piscataway, N.J.) resulting in vector pHR140; pGEX-4T-3 is a GSTexpression vector which contains a multiple cloning site downstream ofglutathione S-transferase which is under the control of the placpromoter. The insertion of the P domain into the pGEX-4T-3 vector wasconfirmed by restriction digest and sequence analysis.

TABLE 9 Oligonucleotides Used in Selection and Characterizationof Aptamers with Binding Affinity to Noroviruses Name Sequence (5′-3′)SEQ ID NO DNA Aptamer AGTATACGTATTACCTGCAGC-(N)₄₀-CGATATCTCGGAGATCTTGC146 Library Sequence FAM-Forward FAM-AGTATACGTATTACCTGCAGC 147Constant Region Biotin-Reverse Biotin-GCAAGATCTCCGAGATATCG 148Constant Region Forward Constant AGTATACGTATTACCTGCAGC 147 RegionReverse Constant GCAAGATCTCCGAGATATCG 148 Region P493*GCACGGATCCTTTTTAGTCCCTCCTACGGTG 156 P494* GGACGCGGCCGCTTATCGGCGCAGACCAAG157 *The underlined bases in P493 and P494 correspond to BamH1 and Not1enzyme sites, respectively.

For expression of the P protein, pHR140 was electroporated into E. coliBL21 cells (Invitrogen, Carlsbad, Calif.) following the manufacturer'sinstructions. Transformants were selected for on LB media containing 100μg/ml ampicillin. A 10 ml overnight culture of cells was diluted 1/10 in2×YT (Yeast extract-tryptone) broth and grown to an OD_(600nm) of0.6-0.9; 0.5 mM IPTG was added to induce expression, and the culture wasleft to grow at 37° C. for 3-4 hr in a shaking (250 rpm) incubator. Thecells were harvested by centrifugation, and the supernatant was removed.The cell pellet was then resuspended in PBS containing 1 mMphenylmethanesulfonylfluoride. The cells were transferred to 2 ml screwcap tubes to which 500 μl of 106 micron acid-washed glass beads wereadded. The protein was extracted by beating for 30 s at maximum speed ina Biospec mini bead beater (Biospec Products Inc., Bartlesville, Okla.)followed by 30 s on ice; this was repeated 3 more times. Thereafter,samples were placed on ice and centrifuged at 18,500×g for 5 min(Eppendorf 5424 microcentrifuge, Eppendorf, Westbury, N.Y.) to removethe glass beads and cellular debris.

The P protein was purified using a GSTrapFF column (GE Healthcare,Piscataway, N.J.) following the manufacturer's protocol with minorchanges. Briefly, the column was equilibrated with 5 column volumes ofbinding buffer (I×PBS; 140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mMKH₂PO₄, pH 7.3). The sample was applied to the column at a flow rate of0.2-1 ml/min. The column was capped and the protein was allowed to bindovernight at 4° C. The column then was uncapped and washed with 5-10column volumes of binding buffer at a flow rate of 1-2 ml/min. Theprotein was eluted in 5 column volumes of elution buffer (50 mMTris-HCl, 10 mM reduced glutathione, pH 8.0) at a flow rate of 1-2ml/min. Confirmation of the P-protein identity was done using Westernblotting and a NV (GI.I) mouse-derived antibody (Abeam, Cambridge,Mass.) that was diluted 1/400 prior to application to the membrane.After washing, the membrane was treated with a 1/5000 dilution ofanti-mouse IgG conjugated to alkaline phosphatase (Sigma Aldrich, StLouis, Mo.), with color development done using BCIP/NBT liquid substrate(Sigma Aldrich, St Louis, Mo.).

Aptamer Selection (SELEX)

Preparation of DNA Library (FAM-Labeling).

An 81-base combinatorial DNA library was constructed (as describedbelow) by producing an 81 bp dsDNA molecule that was labeled at the 5′end with FAM and the 3′ end with biotin. This enabled the preparation ofFAM-labeled ssDNA through biotin coupling at the 3′ end of the 81 bpdsDNA to paramagnetic particles (see below for method). The dilutedaptamer library (10 μM initial concentration) was amplified in a 50 μlPCR reaction containing 1×GOTAQ Buffer (Promega Corp., Madison, Wis.),0.2 mM GENEAMP dNTPs mix (Applied Biosystems, Foster City Calif.), 2 UGOTAQ DNA polymerase (Promega), 500 nM FAM-Forward Constant Regionprimer and 500 nM Biotin-Reverse Constant Region primer (Table 9). ThePCR was performed in a DNA engine Peltier Thermal Cycler 200 (MJResearch/Bio-Rad Laboratories, Hercules, Calif.) using a 3-step thermalprotocol consisting of an initial denaturation at 95° C. for 2 mMfollowed by 30 cycles of 95° C. for 30 s, 55° C. for 30 s and 72° C. for15 s, and a final extension at 72° C. for 5 min.

Separation of FAM-Labeled, Single-Stranded DNA (FAM-ssDNA) During SELEX.

Prior to each round of SELEX, it was necessary to separatesingle-stranded, FAM-labeled DNA from its complementary biotinylatedstrand. To do this, Streptavidin MAGNESPHERE paramagnetic particles(Promega) were washed 3 times in 0.5×SSC buffer before use. TheFAM:Biotin-labeled, double-stranded DNA (dsDNA) was coupled with theStreptavidin MAGNESPHERE paramagnetic particles by incubating at roomtemperature for 30 min with end-over-end mixing. The dsDNA-coupledmagnetic beads were washed 3 times with 0.IX SSC buffer. The FAM-labeledssDNA moieties were separated from the immobilized biotin-labeledstrains by alkaline denaturation in 0.15M NaOH at room temperature for3-5 min. The biotin-ssDNA strands attached to the magnetic beads werecaptured using a Dynal MPC-M magnetic particle concentrator (Dynal A.S.Oslo, Norway). The FAM-ssDNA was removed and the NaOH was neutralizedwith 0.15M HCl. The FAM-ssDNA was then recovered by ethanolprecipitation and resuspended in dH₂O.

SELEX Applied to Norwalk Virus (NV) P Protein.

Prior to SELEX, purified P protein was bound to glutathione sepharose 4B(GS4B) (GE Healthcare, Piscataway, N.J.) following manufacturer'sinstructions. Briefly, the GS4B was spun down at 500×g for 5 min, andthe storage buffer was removed. The GS4B was washed in PBS twice, eachtime centrifuging at 500×g for 5 min, and the final pellet wasresuspended in PBS. The P protein was overexpressed as described above,and the protein was extracted by bead beating. The extracted proteinswere mixed with the washed GS4B and allowed to incubate at roomtemperature for 1 h with end-over-end rotation. The GS4B was washed 3times with PBS at 500×g for 5 min to remove the unbound proteins andstored at 4° C. until required. About 300-500 pmol of ssDNA aptamerswere heated at 90° C. for 10 min and cooled on ice. The cooled aptamerswere added to a 250 μl aliquot of P-protein-bound GS4B and allowed tomix gently at room temperature for 1 h. The GS4B was then washed 3 timeswith PBS at 700×g for 5 min to remove any unbound aptamers. Boundaptamers were eluted from the GS4B with 500 μl elution buffer (50 mMTris-HCL, 10 mM reduced glutathione, pH8.0) and recovered byphenol:chloroform extraction and ethanol precipitation. The aptamer poolwas then regenerated by PCR. Counter-SELEX was performed before round 1of SELEX and after rounds 5 and 10. The initial round of counter-SELEXwas performed with protein bound to the GS4B from E. coli containing thepGEX-4T-3 vector. This removed any aptamers that could bind to the GS4Bor the GST region of the fusion protein. After round 5, thecounter-SELEX was performed using a 20% human stool suspensionpreviously screened negative for HuNoV. After SELEX round 10, thecounter-SELEX was performed with GS4B only.

Identification of Aptamer Sequences

After the final round of SELEX, the dsDNA was regenerated as describedabove; however, unlabeled Forward Constant primer and Reverse Constantprimer were utilized in the PCR reaction. The PCR product was thencleaned using the QiaQuick PCR purification kit following themanufacturer's instructions (Qiagen, Valencia, Calif.). The purified PCRproduct was ligated into the pCR2.1 TOPO vector (Invitrogen, Carlsbad,Calif.) according to the manufacturer's instructions and electroporatedinto E. coli Top10 cells. Transformants were selected for as whitecolonies on LB-Xgal plates containing 50 μg/ml kanamycin. The selectedtransformants were grown overnight, and the plasmid DNA was extractedusing the QlAprep Spin plasmid miniprep kit (Qiagen Inc., Valencia,Calif.). The plasmid DNA was then sent to Genewiz (South Plainfield,N.J.) for sequencing.

Prediction of Aptamer Structural Folding and Characterization of AptamerBinding

The structural folding analysis of the NV and SMV candidate aptamersequences was done using the DNA Mfold online server (found at thewebsite located at mfold.ma.albany.eduno=mfold/DNA-Foldirw-Form). Abinding assay was developed and used to characterize binding affinitiesof candidate aptamers to NV.

Norwalk:Aptamer Binding Assay

This assay is similar to the two-site binding assay described above.Specifically, mouse GI.I (NV) antibody (Abcam) was biotinylated using anEZ-Link Sulfo-NHS-LC-Biotin and biotinylation kit according to themanufacturer's instructions (Pierce Biotechnology, Inc. Rockford Ill.).Streptavidin MAGNESPHERE paramagnetic particles (Promega) were washed asdescribed above in 0.5×SSC and bound to the biotinylated GI.I antibody.After washing, a 50 μl aliquot of 20% NV stool suspension was added tothe antibody:bead complex, and the suspension was mixed for 1 h at roomtemperature with end-over-end rotation. The bead:antibody:virus complexwas then washed 3 times with PBS to remove unbound virus and fecalmaterial. The aptamer was heated at 90° C. for 10 min, cooled on ice, 1nM of the aptamer was added to the bead:antibody:virus complex, and thesamples were mixed for 2 h at room temperature with end-over-endrotation. The bead:antibody:virus:aptamer complex was washed 3 timeswith PBS to remove any unbound aptamer. This was followed by a heatrelease to elute the aptamers from the surface of the virus. Therecovered suspension was used in a SYBR green real-time PCR reactiontargeting the aptamer. The real-time PCR assay was performed in aSmartCycler (Cepheid, Sunnyvale Calif.) using 200 nM unlabeled ForwardConstant Region primer, 200 nM unlabeled Reverse Constant Region primerand IX SYBR green master mix (Applied Biosystems, Foster City Calif.).The PCR protocol was as follows: 95° C. 10 min followed by 95° C. for 15s, 55° C. for 15 s, and 72° C. for 15 s for 40 cycles.

Aptamers with Binding Affinity to the Norwalk Virus P Protein

After five rounds of SELEX and two rounds of counter-SELEX (done usingthe pGEX-4T-3 vector only, and norovirus-negative stool), a total of 22candidate aptamer sequences were identified (representative sequencesshown in Table 10). Several of these were chosen for furthercharacterization, based in part on frequency in which that aptamer wasidentified (e.g., aptamer NV 1-1) and unique structure (e.g., aptamerNV1-24). The free energies (dG) of NV 2-1, NV 2-9, NV 2-3, NV 1-1, NV1-24, and NV 1-15 were −13.09, −13.11, −5.82, −9.94, −5.05, and −5.05Kcal/mol, respectively.

TABLE 10 Representative Aptamer Sequences Obtained from 5^(th) and10^(th) Rounds of SELEX for Norwalk Virus (NV) Round of SEQ # AptamerSELEX Random Region Sequence ID NO Repeats Identifier  5GGGGTGGTGCCGGAGTGGGGTGGCGGTGCGGATTCCCTGGCTATGCC 41  8 NV 1-1  5TGGGGGGTGGTGCGGTGTGTGACAAGGGAGCATAGCCGGGGGCCAGT 42  2 NV 1-15  5TTGGTTGGTGCTCGCTGTAAGGTTAACACCGTCTAATCGGGACCGT 43  2  5GGGGAGCTCGTGGGTAGAGTGGGGCCGGGGTGTGGTATAGTGCGGCC 44  1  5TCGTCCTAGTGTGGGATATAGCTATGAAATCAACTTTCCC 45  1  5TGTAGGGAGTTGTTACATCGGCACTGGTCTGTTGAATTCT 46  1  5GGGGAATGTTCTTGTGGCCTACCGGGGAGTGGCCTTTATGTCCCTT 47  1  5GGTGGGGGGGTGCGGCATGTGGAGGCGGCGGGCAGGAGGGGGACAGTG 48  1  5TGGGGGGGAGGGGAGATGGGTGGCGGTGGTGGGGCTTAGGGCTATCC 49  1  5GTGGACGGTAGTCGTTGTGGGGCGCGGTGCGGGGGGGTTCGGGCGTG 50  1  5TGGGAAAGGGAAGTGTGGGCAGGGGAGGGAGGGGGGTGGCTACATCA 51  1  5TAGGGCAATATGTTAGTTAGGCGACTTGCTTAGACTACTG 52  1  5TTTGATGGTGCGGTGGCTTACATATGCGTTCTACATTGCGTTCGG 53  1 NV 1-24 10TAATTGTGTGTCGCAGCATGGTGGTGCCGGGCCTTGCATCCACCTTCGG 54 14 10TGGGGGGGGGGGCTGCTAAAGGTTTGTGGAGGGTTAACATGTACCTCCCC 55  7 NV 2-9 10TGGGAAAGGGAAGTGTGGGCAGGGGAGGGAGGGGGGTGGCTACATCA 56  9 NV 2-3 10TGGATGGGGTGATGCTGGGTGGAAGAGGGGGCCGGACCCGCCGTCCGTG 57  2 10TTGGTTGGTGCTCGCTGTAAGGTTAACACCGTCTAATCGGGACCGT 58  2 10TGGGGGGTGGTGCGGTGTGTGACAAGGGAGCATAGCCGGGGGCCAGT 59  4 10GGGGTGGTGCCGGAGTGGGGTGGCGGTGCGGATTCCCTGGCTATGCC 60  3 10TGGGGGGTGGTGCGGCATGTGGAGGCGGCGGGCAGGAGGGGGACAGTG 61  1 10GGTGGGGGTGTGACCGGTGTGAGTCCGGTCCCGACGCGTGGATTCGG 62  1 NV 2-1 10TGGGAATAGGGAAGTGTGGATGAGTTCTGAGGATACCACGCCTTACCC 63  1 10TTGAATGGTGGCAGTTGTTGAGGGGAGGTGTCGGGGGGGGCGTTCGT 64  1 10TGGGAAAGGGGGAGAGTTGTGTGGCGAGCGTTGGACGGTGTGCCCCC 65  1

The norovirus:aptamer binding assay was utilized to confirm thataptamers NV 1-1 and NV 1-24 were able to bind to the surface of the NV.Using this protocol, we were able to confirm that Aptamers NV1-1 and NV1-24 did bind to NV, as evidenced by successful amplification (with lowCt value) using the SELEX constant primers, and a corresponding shift inT_(m). In the absence of virus, the aptamers were not amplified, and theassociated T_(m) values corresponded to those observed for primer-dimer,not target-specific PCR amplification (Table 11).

TABLE 11 Real-Time RT-PCR Data Obtained for Norovirus:Aptamer BindingAssay Melting Temperature Sample PCR Product Seen (T_(m)) NV 1-1 withvirus + (Ct 13.84) 84.75 NV 1-24 with virus + (Ct 16.80) 79.25 NV 1-1without virus − 62.5 NV 1-24 without virus − 62.65 No template control −63.44

Example 5: Multiple Graphene Oxide (GO)-SELEX for Efficient Screening ofAptamers for Norovirus Genogroup GI

Graphene and its derivative graphene oxide (GO) are promising moleculesfor a variety of biotechnological uses, with features such asfluorescence quenching, ability to protect biomolecules from enzymaticcleavage, ssDNA adsorption, and desorption of ssDNA in the presence of aspecific target (Nguyen et al., Chem. Commun. (Camb) September 18;50(72):10513-10516 (2014); Chen et al., J. Agric. Food Chem.62(42):10368-10374 (2014); Park, Chem. Commun. (Camb) February 18;48(15):2071-2073 (2012)). The use of GO-Selex method for virus particlesoffers a simple to use, cost effective and immobilization-free platformfor screening of aptamers that bind to their target with high affinityand specificity. Herein, we report a simple aptamer screening method fora group of norovirus GI VLPs using graphene oxide (Multi-GO-Selex)without immobilizing targets.

Three hundred picomoles of the pool of aptamers were incubated with 400μg of GO in 1 ml of 1×PBS buffer for 45 min at room temperature. Theunadsorbed aptamers were discarded in the supernatant by centrifugationat 10,000×g, 7 min. The pellet with the bound aptamers was incubatedwith 5 different GI VLPs (GI.1, GI.4, GI.6, GI.7 and GI.8) as targetsfor specific desorption of the aptamers with a concentration of 10¹³particles. After 2 h incubation at RT, the pool of aptamers bound toVLPs were recovered in the supernatant by centrifugation at 10,000×g,for 7 min. The pool was amplified and purified as described previously(Escudero-Abarca et al., PLoS One 9:e106805 (2014). This constituted oneselection round.

After four rounds of GO-Selex, the enriched pool was cloned and 9 cloneswere sequenced. Three copies were found to have identical copies(AP6-GI), thus a total of 6 candidates were obtained. The structuralfolding analysis of GI candidate aptamer sequences was done using theDNA Mfold online server. The secondary structures predicted at roomtemperature (RT) in the presence of 137 mM NaCl which is theconcentration of the salt in the buffer PBS used during the Selexprocess, showed free energy (dG) values of dG=−10.67 (AP-GI); dG=−8.74(AP2-GI); dG=−5.78 (AP3-GI); dG=−9.26 (AP4-GI); dG=−6.98 (AP5-GI) anddG-10.99 (AP6-GI). See Table 12 and FIGS. 23-28.

TABLE 12Aptamer Sequences Obtained from 4^(th) Round of SELEX for GI-VLPs #Repeats Aptamer Random Region Sequence in Pool SEQ ID NO AP1-GICAGGATTAGTCATGGAATAGCCGACGATCATGACCCATTG 1 176 AP2-GICAGCGAAGGGACAGTTCTACGAATGTGAACATGAGGTAGC 1 177 AP3-GITGTTGGATTGATCCTAATTACGGATATTTACACGAATG 1 178 AP4-GITCACGGCGAATCGAAGGGACGCCGCGAAGTGTACCAAGTG 1 179 AP5-GICTGGTCCAGTCAAGGGGATTAGATGAGGGGTAATGGAGAG 1 180 AP6-GICCGAGTAGGGCCGGTCGTCACGGAGAAGCAGGGTGAGCGT 3 181

Common sequence identification of the random regions of the aptamers inthe sequence pool was conducted using the MEME server for analysis ofonly the input aptamer strands with a minimum motif length of 6 bases(Bailey & Elkan, Proc. Int. Conf. Intell. Syst. Mol. Biol. 2:28-36(1994); Bailey et al., Nucleic Acids Res. 37:W202-W208 (2009)). Motifsof at least 6 bases with no more than two mismatches were selected ascandidate motifs. The motifs are provided in Table 13.

TABLE 13 Motifs for GI SELEX Round 4 Sequences Motif Sequence SEQ ID NOAptamers 15 ACGAATG 182 AP2-GI, AP3-GI 16 ACGGAT 183 AP3-GI* 17CGAAGGGAC 184 AP2-GI, AP4-GI 18 CGAAGTGTAC 185 AP4-GI 19 GGNGAGCG 186AP5-GI, AP6-GI 20 NGTNGG 187 AP3-GI, AP6-GI 21 NGGTNG 188 AP2-GI, AP6-GI22 GGATTAG 189 AP1-GI, APS-GI 23 GGAATAG 190 AP1-GI *Occurs twice insame aptamer.

Example 6: Discriminating Between Infectious and Non-InfectiousNorovirus Capsid Integrity and Functionality

The inability to culture HuNoV prevents the direct detection ofinfectious virus by molecular methods such as RT-qPCR. The reason forthis is that naked or even partially degraded RNA may persist in theenvironment long after inactivation of the viral capsid or whole genome,and hence could be amplified by RT-qPCR after the virus has lost itsability to infect a host cell. This is of particular concern in foodsand water, as various physical and chemical methods (e.g., heat, highhydrostatic pressure, ultraviolet light, ozone, chlorine, ionizingradiation, etc.) are commonly used to inactivate microorganisms ofconcern. In the case of viruses, the presence of a positive molecularamplification signal may erroneously imply the failure of the method toinactivate HuNoV.

In an effort to use molecular methods to aid in predicting viralinfectivity, some have capitalized on specific properties of aninfectious virus. One of these is the integrity of the viral capsid,which should theoretically protect the viral RNA from damage ordegradation. The viral capsid is frequently the first to be degradedwhen viruses are subjected to inactivation methods. One approach in thisregard is to precede RT-qPCR with a proteinase K and/or RNase digestiontreatment, the idea being that partially degraded capsids will be fullydegraded by the former enzyme, while exposed RNA will be degraded by thelatter. Another approach is to precede RT-qPCR by a virus capture stepusing an immobilized ligand with affinity to HuNoV. Such ligands caninclude antibodies, histo-blood group antigens (HBGAs; the putativereceptors/co-factors for HuNoV), or porcine mucin. Purportedly, thesemethods are intended to discriminate infectivity status based on theintegrity of the viral capsid assuming that non-intact capsids would notbe able to bind to the appropriate ligands. Further information on thesetypes of methods can be found in Knight et al., Crit. Rev. Microbiol.39:295-309 (2012). We maintain that aptamers could be a candidate ligandin these types of assays.

The most commonly used ligands for virus capture prior to RT-qPCR isHBGAs or porcine gastric mucin (which contains some HBGAs). Thesereceptors are fairly costly, require purification from animals, andcannot be easily functionally modified with chemical groups. BecauseHuNoV binding is strain-specific, no HBGA broadly reacts with all HuNoVstrains, and some HuNoV do not bind any HBGAs (see Murakami et al., PLoSOne 8(6):e66534; Donaldson et al., Immunol. Rev. 225:190-211 (2008)).This further complicates detection and diagnosis of HuNoV. Despitereports of some broadly reactive antibodies, no antibody has beenidentified capable of binding all HuNoV strains (see Huang et al., Prot.Eng. Design & Sel. 27(10):339-349 (2014); Kou et al. Clin. VaccineImmunol. 22(2):160-167 (2015); Shiota et al. (2007)). Nucleic acidaptamers are an interesting alternative but have not yet been used inthis application. Multiple aptamers have been generated againstnoroviruses, some of which are broadly reactive (see Beier et al., FEMSMicro. Let. 351(2):162-169 (2014); Escudero-Abarca et al., PLoS One9(9):e106805 (2014); Giamberardino et al., PLoS One 8(11):e79087 (2013);Moore et al., J. Biotech. Accepted Manuscript). In this study, weinvestigated whether loss of aptamer binding correlates with loss ofHBGA or antibody binding for heat-treated HuNoV capsids as a potentialproxy for estimating capsid integrity/functionality.

Materials and Methods Virus-Like Particles (VLPs)

VLPs consisting of the assembled recombinant HuNoV capsid proteins ofdifferent HuNoV strains were used to investigate HuNoV capsid integrity.Specifically, GII.2 Snow Mountain (SMV), GII.4 Houston (HOV), and GII.4Sydney (SDV) strains from human norovirus genogroup II were generouslyprovided in purified form by R. Atmar (Baylor College of Medicine,Houston, Tex.). VLPs were stored in buffer at >1.0 mg/ml concentrationand 4° C. until use.

Nucleic Acid Aptamers

Biotinylated versions of two ssDNA aptamers previously reported to bebroadly reactive to HuNoV strains were selected for use in the study.One aptamer, SMV-19, was generated with a FAM label against infectiousSMV from patient stool isolates (see Escudero-Abarca et al., PLoS One9(9):e106805 (2014)). The other aptamer, M6-2, was generated with abiotin label against the P domain of a GII.4 2006b HuNoV strainexpressed in Escherichia coli (see Moore et al., J. Biotech. AcceptedManuscript).

Heat Treatment of VLPs

VLP suspensions were diluted to 50 μg/ml in 1× phosphate-buffered saline(PBS; pH 7.2) or 10 mM HEPES (pH 7.4) for plate assays and electronmicroscopy, respectively. Samples were placed in 15 μl aliquots in 0.2ml PCR tubes and heated using a T100 Thermal Cycler (Bio-Rad, Hercules,Calif.) at different temperatures [60° C., 65° C., 70° C., 75° C., 80°C.] for 1 min and at selected temperatures [63° C., 65° C., 68° C., 70°C., 72° C.] for 2.5-100 min, depending upon strain and experimentaldesign. In other experiments, VLPs were treated at 80° C. for 5 min(completely denatured) or left untreated for use as negative andpositive controls, respectively.

Immediately after heat treatment, samples were placed in a DNA Engine(PTC-200) Peltier Thermal Cycler (MJ Research, Hercules, Calif.) runningat 4° C. for 5 min to cool. For plate assays. VLP aliquots were brieflycentrifuged and diluted in 1×PBS to 3 μg/ml. One hundred μ1 of thedilutions were then applied to Costar 3591 medium-binding polystyrene96-well plates (Fisher, Pittsburgh, Pa.) overnight at 4° C. with lightshaking using an orbital shaker. Plates were processed by aptamer, HBGA,and/or antibody binding assays as described below.

Enzyme-Linked Aptamer Sorbent Assay (ELASA)

Aptamer binding to treated VLPs was probed using a previouslyestablished assay (see Escudero-Abarca et al., PLoS One 9(9):e106805(2014); Rogers et al., J. Clin. Micro. 51(6):1803-1808 (2013)). Briefly,the wells containing the VLPs or PBS were blocked with 200 μl of 5% skimmilk in PBS-Tween 20 (0.05% v/v; PBST) with a 10 nM mix of unrelated PCRprimers (Listeria monocytogenes primers hlyQF/R and L23 SQF/R)(Rodriguez-Lazaro et al., FEMS Microbiol. Lett. 233:257-267 (2004)) for2 h at room temperature with shaking. The plates were washed thrice with200 μl of PBST and then incubated with 100 μl of 1 uM biotinylated M6-2or SMV-19 aptamer for 1 h. Plates were washed 4 times with PBST andincubated with 100 μl/well of a 1 mg/ml streptavidin-horseradishperoxidase diluted 1:5,000 (v/v; Invitrogen, Carlsbad, Calif.) in PBSfor 15 min. Residual conjugate was removed with 3 wash steps of PBST andplate signal developed with 100 μl/well of the3,3′,5,5′-Tetramethylbenzidine (TMB) microwell peroxidase substratesystem (KPL, Gaithersburg, Md.) per manufacturer's instructions. Signalwas allowed to develop for 2-7 min before reactions were stopped withthe addition of TMB stop solution (KPL). The absorbance at 450 nm wasthen recorded using a Tecan Infinity M200pro microplate reader (TecanGroup Ltd., Männedorf, Switzerland). For all plate-based assays, aminimum of two wells per treatment per plate and three replicate platesfor each treatment experiment were conducted. Signal development timewas kept consistent within 30 seconds for each replicate.

ELISA-Like Histo Blood Group Antigen (HBGA Binding) Assay

Binding of heat-treated VLPs to HBGAs was observed simultaneously and inparallel to aptamer binding of heat-treated VLPs to aptamers. The HBGAbinding assay has been previously reported (see Manuel et al., Appl.Environ. Micro. In Press) for observing capsid degradation and issimilar to the ELASA except for the following: wells were washed twicewith PBST after blocking and incubated with 30 μg/ml biotinylated bloodtype A HBGA (#01-032, GlycoTech, Gaithersburg, Md.) in 100 μl blockingbuffer for 1 h. Wells were subsequently washed thrice and the signaldetermined as above using the streptavidin-horseradish peroxidase asabove. Signal was developed for 10-20 min before stopping and reading at450 nm.

Enzyme-Linked Immuno-Sorbent Assay (ELISA)

For the GII.4 Sydney VLPs, aptamer and HBGA binding was compared toantibody binding for heat treatments using a previously described ELISAmethod (see Hansman et al., J. Virol. 86(7):3635-46 (2012); Koho et al.,J. Virol. Meth. 179(1):1-7 (2012)). Briefly, VLPs were blocked for 2 has above and washed thrice with PBST. Wells were then incubated with 100μl of 0.137 μg NS14 antibody (kindly provided by R. Atmar, BaylorCollege of Medicine, Houston, Tex.) (see Kitamoto et al., J. Clin.Micro. (2002)) in 0.1% skim milk-PBST for 1 h. Plates were washed thricewith PBST and wells incubated with 0.1 μg goat anti-mouse-horseradishperoxidase antibody (#62-6520, Invitrogen) in 100 μl of 0.1% skim milkfor 1 h. Wells were washed thrice with PBST and signal developed asabove with the TMB substrate system for 1-5 minutes before reactionswere stopped and read.

Plate Data Analysis

For plate analysis, absorbances of negative control wells for eachligand seeded with completely heat denatured VLP (80° C., 5 min) weresubtracted from sample well absorbances to remove nonspecific andprimary sequence-based ligand interaction. Negative-adjusted absorbancesof the samples for different temperature/time points were then taken asa percentage of the negative-adjusted absorbances of untreated positivecontrol VLPs. At least two wells per temperature-time point per plateand three replicate plates were performed. For each VLP degradation at aspecific temperature, a range of times where the approximate linearportions of the aforementioned signal degradation based on the HBGAbinding loss were selected and linear trendlines were drawn andequations with R² values calculated using Microsoft Excel. Slopes forthe lines for each ligand at the same selected time points—whichindicates the apparent rate of signal loss—were calculated for eachplate replicate. Average rates of apparent signal loss with standarddeviation were calculated and unpaired t-tests with Welch correctionwere conducted comparing the three ligands for each temperature usingGraphPad Prism version 5.0d (San Diego, Calif.). To estimate theapparent percentage of signal attributable to VLP proteinsequence-dependent versus confirmation-dependent binding for all threeligands, three replicates of GII.4 Sydney VLP plates were completed.Instead of subtracting the absorbance values of denatured capsid (80° C.for 5 min), the absorbance value of a no VLP well (accounting for signaldue to the plate apparatus) was subtracted. The subtracted values ofpositive control (untreated) and completely denatured VLP (80° C., 5min) were used in the equation below to estimate the percentage ofsignal attributable to completely denatured capsid (sequence-dependentbinding): [(80° C. 5 min wells average absorbance)−(No VLP wells averageabsorbance)]/[(Untreated VLP wells average absorbance)−(No VLP wellsaverage absorbance)]*100 The apparent percentage of ligand binding tocompletely denatured capsid for each replicate plate were then averagedand their standard deviations determined using Microsoft Excel 2013.

Transmission Electron Microscopy

In order to confirm VLP degradation and observe morphological changesfor VLPs after different degrees of heat treatment, TEM images weretaken corresponding to different heat treatments relevant to thoseconducted for binding assays. VLPs were heat treated and cooled at 50μg/ml in 10 mM HEPES (pH 7.4) as described above, and then applied tocarbon-coated nickel grids (Ladd Research, Williston, Vt.) for 10 min.Samples were stained with 2% uranyl acetate (SPI Supplies, West Chester,Pa.) for 45 seconds, dried in a desiccator, and viewed using a JEOL 1210transmission electron microscope (JEOL-USA Inc., Peabody, Mass.) at 80kV and 50,000×.

Results

Behavior of Two Different Aptamers with Snow Mountain Virus (SMV)

Initial tests were done to compare the binding behavior of twopreviously selected aptamers to heat-treated VLPs. Specifically, aptamerSMV-19, selected against whole virus (see Escudero-Abarca et al., PLoSOne 9(9):e106805 (2014)), and M6-2, produced against the P domain of aGII.4 2006b HuNoV strain (see Moore et al., J. Biotech. AcceptedManuscript) were used. Snow Mountain Virus (SMV; representing a GII.2strain) was exposed to various temperatures (65-80° C.) for one minfollowed by detection using ELASA. VLP binding to the aptamers wasvirtually unaffected by treatments at 65° C. and 70° C. Some signal lossoccurred at 75° C., and nearly all signal (˜90%) was lost with treatmentat 80° C. for one min. Both aptamers behaved nearly identically in termsof degree of signal loss at each temperature treatment (FIG. 19).Corresponding EM data showed gradual changes capsid structure and thenumber of intact VLPs at intermediate temperatures, and complete loss ofcapsid integrity when ELASA signal was lost (<90% signal reduction).

Further Observation of SMV VLP Degradation Using Aptamer M6-2

To further investigate aptamer binding with SMV VLPs over moreintermediate treatment intensities, temperatures of 70° C. and 72° C.were used with differing exposure times (0-52.5 min). Samples were thentested using ELASA and in some instances, TEM (e.g., 72° C. treatment).At 70° C., ELASA signal loss began within 7.5-15 min of heating, withcomplete signal loss (>90%) observed after 52.5 minutes (FIG. 20B). At72° C., initial signal loss was observed after 2.5 min of heating, and amajority of signal (>85%) was lost after over 17.5 min (FIG. 20A). EMdata resembled that described above (data not shown).

Comparison of Aptamer and HBGA Binding to Houston Virus (HOV) VLPs

In the next set of experiments, aptamer and HBGA binding were comparedas applied to heat-treated HOV VLPs. HOV VLPs were chosen for this workas they have been reported to bind well with both type A HBGA andaptamer M6-2 in direct plate assay formats (see Manuel et al., Appl.Environ. Micro. In Press; Moore et al., J. Biotech. AcceptedManuscript). Initially, VLPs were exposed to temperatures ranging from60-80° C. for one min followed by ELASA or HBGA-binding assay. ParallelEM was done for each sample. Decreases in ELASA signal occurred between65 and 75° C., with HBGA and aptamer curves similar although notidentical (FIG. 17). EM revealed slight morphological changes fortreatment at 75° C., and VLP integrity was completely lost afterexposure to 80° C. for 1 min.

When HOV VLPs were exposed to various time-temperature combinations (63°C. and 65° C. for up to 30 min), both aptamer and HBGA binding signalswere quite similar (FIGS. 18A and 18B). In most cases, there were nostatistically significant differences (p>0.05) in signals at any onetime-temperature combination. For 63° C. over the range of 0-15 min,rates of degradation of VLPs measured by binding assay using eitherligand were not statistically significantly different (Table 14).Notable morphological changes in the form of disfigured capsids wereobserved in increasing proportion as treatment time was lengthened,although intact capsids could still be observed after most of the HBGAor aptamer signal was lost.

TABLE 14 Degradation Rates of Different Human NoV VLPs as Measured byThree Different Ligands Treat- ment Degradation Tem- Times Rate peratureUsed (- VLP Ligand (° C.) (min) % Signal/min) R² SDV Aptamer M6-2 650-75 1.20 ± 0.03 0.90 ± 0.04 SDV HBGA Type A 65 0-75 1.23 ± 0.02 0.97 ±0.01 SDV Antibody NS14 65 0-75 0.76 ± 0.09 0.87 ± 0.07 SDV Aptamer M6-268   0-17.5 5.55 ± 0.13 0.92 ± 0.01 SDV HBGA Type A 68   0-17.5 5.68 ±0.10 0.96 ± 0.01 SDV Antibody NS14 68   0-17.5 3.24 ± 0.24 0.85 ± 0.04HOV Aptamer M6-2 63 0-15 5.96 ± 0.32 0.97 ± 0.01 HOV HBGA Type A 63 0-155.87 ± 0.13 0.97 ± 0.02 HOV Aptamer M6-2 65 0-6  16.20 ± 1.00  0.98 ±0.02 HOV HBGA Type A 65 0-6  16.51 ± 2.97  0.96 ± 0.01

Comparison of Aptamer, HBGA, and Antibody Binding for GIL4 Sydney VLPs

Aptamer, HBGA, and antibody binding were compared for GII.4 Sydney (SDV)VLPs exposed to 65° C. for up to 100 min, and 68° C. for up to 25 min. Abroadly reactive GII antibody, NS14 was used in these studies (seeKitamoto et al., J. Clin. Micro. (2002)). Gradual signal reduction withincreasing exposure time to heat was observed for all binding assays,and complete loss of signal (>90%) occurred at the maximum exposuretime. However, signal reduction, when assessed by HBGA binding assayoccurred earlier (beginning almost immediately after initial exposure toheat), followed by that for aptamer and then antibody (FIGS. 21A and21B). The rates of ELASA signal loss generally mimicked that of the HBGAbinding assay, whereas the data for the antibody binding assay weredifferent. This is supported by the difference in the apparent ratesobserved over the 0-75 min range at 65° C., as aptamer and HBGA signaldegradation rates were not significantly different, but the NS14antibody signal degradation rate was significantly different from bothHBGA and aptamer M6-2 (p<0.05) (FIG. 21B). A similar result was observedfor the 68° C. treatment (Table 14).

Degree of Sequence—Versus Conformation-Dependent Binding of Ligands

To evaluate the ability of each ligand to bind to completely destroyedcapsid, GII.4 Sydney VLPs were completely inactivated by treatment at80° C. for 5 min and assayed using HBGA, antibody, and aptamer bindingassays. Results were expressed as the ratio (%) of ligand binding signalof the heated VLPs to the untreated control VLPs. Aptamer M6-2(1.98±1.27%) and HBGA (0.50±1.15%) bound very poorly to completelydenatured GII.4 Sydney VLP (<3% of positive control signal), butantibody NS14 displayed a much higher degree of binding (26.35±3.85% orpositive control signal) (FIG. 22).

Discussion

In this study, the ability of ssDNA aptamers to bind various HuNoV VLPs,which were partially or completely inactivated by heat, was assessed.The performance of the aptamers was directly compared to that of type AHBGA and a HuNoV antibody, as these are common ligands used for virusbinding and to aid in discrimination of virus infectivity status bypreceding RT-qPCR with a specific virus capture step. In this way,ligand binding can serve as a proxy for HuNoV capsid integrity.Representative VLPs were treated at time-temperature combinations thatproduced varying degrees of capsid disruption and/or destruction. Whencomparing two aptamers to one another, both performed equivalently forheat-treated SMV VLPs, suggesting that they can be used interchangeably.The similar performance of the two aptamers may be a function ofsimilarities in aptamer size, structure, binding affinity, and/orvirus-ligand binding site, among other possible factors.

In the second phase of work, the binding of heat-treated GII.4 Houston(HOV) VLPs to aptamer and type A HBGA were compared. Capsid degradationprofiles for HOV exposed to heat were very similar when comparingaptamer M6-2 and HBGA binding results, suggesting that aptamer M6-2might be an alternative to HBGA for use in studies in whichconsideration of capsid integrity is necesary.

When aptamer, HBGA, and antibody binding were compared for heat-treatedGII.4 Sydney VLPs, a relationship between treatment time-temperature andligand biding was observed for all three ligands. The rates of aptamersignal loss as a function of time-temperature generally mimicked thoseof HBGA, whereas the rate of signal loss for the antibody-based assaywas significantly different. This would imply that both aptamer M6-2 andHBGA binding to GII.4 Sydney VLPs strongly depends on higher orderprotein conformation.

Indeed, a high degree of conformational dependence has been demonstratedusing heat denatured Norwalk Virus VLPs and synthetic HBGA (seeHarrington et al., J. Virol. 76(23):12335-12343 (2002)) and has beenobserved for other treatments (see Manuel et al., Appl. Environ. Micro.In Press), as HBGAs and receptor binding have been found to approximatecapsid functionality and infectivity for heat-treated virus (see Danchoet al., Intl. J. Food Micro. 155(3):222-226 (2012); Hirneisen & Kniel,J. Virol. Meth. (2012)). Additionally, similar observations of aptamerdependence on nondenatured target protein have been observed (seeTakemura et al., Exp. Bio. Med. 231(2): 204-214 (2006)). Others havefound that the nature of aptamer binding interactions with targets canbe notably different than interactions with protein ligands likeantibodies (see Hermann & Patel, Sci. 287:820-825 (2000)). Collectively,this literature is consistent with our finding that that the aptamer andHBGA bound very poorly to completely denatured GII.4 Sydney VLP, butantibody NS14 displayed a much higher degree of binding to the denaturedVLPs.

Comparatively speaking, different VLPs showed different thermalinactivation profiles. Overall, SMV VLPs were most heat resistant,followed by GII.4 Sydney VLPs and then HOV VLPs. The fact that twodifferent GII.4 VLPs (NOV and Sydney) have different thermalinactivation profiles is interesting as both represent epidemic strains,albeit having emerged at different times. Strain-specific differences incapsid structure likely mediate this observation. These results havepractical significance as thermal inactivation data collected for onehuman NoV strain may not necessarily reflect the behavior of another.

TEM images of capsid integrity roughly correlated with loss of HBGAbinding at broader treatments (such as the different temperaturetreatments), but capsids appeared to retain structure after loss ofcapsid integrity when observed at more gradual heat treatments (such asset temperatures for different times). This is consistent with theobservations of other studies (see Lou et al., Appl. Environ. Micro.78(15):5320-5327 (2012); Manuel et al. Appl. Environ. Micro. In Press)and is somewhat expected given the highly conformation-dependent natureof HBGA binding discussed above. This phenomenon has implications forproper quantification of infectious human NoV particles in detection, asRNase pre-treatment of samples prior to RT-qPCR are not likely toaccount for particles that remain intact but have lost HBGA bindingfunctionality. Shortcomings in the ability to properly predictinfectious human NoV particles have been discussed elsewhere (see Mooreet al., Annual Rev. Food Sci. & Tech. 6(1):411-433 (2015); Knight etal., Crit. Rev. Micro. 39(3):295-309 (2013)).

Although the plate-based assays allow for direct comparison of capsidintegrity estimation, absolute quantification (i.e., log₁₀ reductions)is not possible due to assay design, relatively high assay detectionlimits, and variations in VLP solution purity. However, the plate-basedmethods do allow for relative quantification, and results were supportedby TEM, providing additional support to the validity of the comparisonsmade in this study.

This study demonstrated that loss of aptamer binding correlates withloss of HBGA binding (and to some extent, loss of antibody binding) toHuNoV capsids subjected to sublethal or lethal damage by heat. HBGAs andantibodies are frequently used to capture intact HuNoV prior todetection using RT-qPCR. It is believed that a ligand binding stepexcludes non-binding (and hence, non-infectious) virus particles, andsubsequent RT-qPCR is more discriminatory for infectious virus. Inshort, ligand binding is used as proxy for estimating capsidintegrity/functionality. The results presented here show suggest thatsome virus particles remained intact after non-lethal heat treatment,but capsid regions with which the aptamer-virus interaction occured mayhave been altered. Hence, capsid integrity/functionality could have beenimpacted. Such a phenomenon would likely impact virus infectivity, inwhich case aptamer binding could be a promising means by which toassesss HuNoV infectivity prior to detection using molecularamplification. However, in the absence of a HuNoV cultivation method,this cannot yet be definitively proven. A study similar to this oneusing a cultivable surrogate to which an aptamer has been produced,would be a logical next step.

HuNoV Nonstructural Polyprotein

In addition to the requirement of capsid integrity and functionality,the correct expression and presence of the HuNoV nonstructuralpolyprotein (encoded in ORF1) is required for HuNoV to be infectious.ORF1 is first translated as a polyprotein that cleaves into sixdifferent nonstructural proteins. One of these proteins, the VPg, iscovalently bound to the 5′ end of HuNoV genomic and subgenomic RNA, andit is suspected to be involved in translation of viral RNA (Daughenbaughet al., Virol. 1 3:33 (2006); Goodfellow et al., EMBO Rep. 6:968-972(2005)), as well as potentially serving a role in initiatingtranscription (Belliot et al., Virol. 374:33-49 (2008); Machin et al.,J. Biol. Chem. 276:27787-27792 (2001)). It has been demonstrated thatthe VPg is required for cultivable surrogates of HuNoV to be infectious(Guix et al., J. Virol. 81:12238-12248 (2007)). Additionally, HuNoV VPgis under less selective pressure than the viral capsid, which may makeit a more suitable target for the creation of a broadly reactive ligand.

Because the VPg is required for replication of the virus, it is expectedthat it must also be functional (in addition to the capsid) forinfectious viruses. Therefore, the previously discussed capsid integrityand binding functionality methods and RNase treatment methods could becombined with an aptamer-based capture step targeting the intact VPg inan effort to discriminate between infectious and non-infectious virus.This might be called an “integrated” method to discriminate virusinfectivity status. Because of the aforementioned advantages ofmodification for aptamers, this approach can be amenable to amicrofluidic device.

Aptamers targeting both HuNoV GI.1 Norwalk Virus (NV) and Tulane Virus(TV) VPgs were created using the same process used for the GII.4 Pprotein. This approach was taken so that the behavior of aptamerstargeting HuNoV VPg could be compared directly to those targeting theVPg of a cultivable surrogate virus (TV), the latter of which would benecessary to make direct correlations between the proposed VPg-basedassay and virus infectivity. Additionally, the fact that TV also bindsHBGAs means that it would be possible to combine ligand-based viruscapture (using HBGAs) with aptamer-mediated VPg capture in an effort toproduce a truly “integrated” approach to discriminating virusinfectivity status.

For the cloning and expression of VPgs, Norwalk Virus (GI.1; NV)clinical stool specimens were used. Tulane Virus (TV), a novel surrogateof norovirus, was cultured as previously reported and a lysatecontaining viral capsid was obtained. The fact that aptamers werecreated against HuNoV and a cultivable surrogate mean that infectivityassays can be validated and developed.

Genomic RNA from both viruses was extracted using a viral extraction kit(Qiagen), reverse transcribed using the Ambion RETROScript kit with 5 μMof primers designed to the VPg region of the cDNA, and product cleanedwith Qiagen Qiaquick kit. Primers for NV were taken from previouslyperformed work (Daughenbaugh et al., EMBO J. 22:2852-2859 (2003)) andare listed in Table 15 below. TV primers were designed using the genomeand cleavage sites estimated by Farkas et al., J. Virol. 82:5408-5416(2008) and are also listed in Table 15 below. The VPg sequences wereamplified by PCR, product cleaned using Qiagen Qiaquick kit, anddigested with EcoRI and Xhol restriction enzymes. Sequences were ligatedinto digested pGEX 4T-1 plasmid vector using the Epicentre Fast-Link kitat a 2:1 insert:vector ratio then electroporated into E. coli BL21 cells(Lucigen E. CLONI electrocompetent cells). Successful transformants wereconfirmed by sequencing (Genewiz Inc.). VPg was overexpressed bydiluting an incubation of overnight cells 1:10 into fresh2×YT-ampicillin broth and incubated at 37° C. with shaking untilOD₆₀₀=0.6-0.9, after which IPTG was added to a final concentration of 1mM. Samples were left to incubate overnight with shaking at 25° C. Thecells were then pelleted, and lysate was made using a bead beater withacid-washed beads (Sigma). Lysate was divided into one-time use aliquotsand stored at −80° C. for use in the same SELEX process as described forthe P Protein. Eight rounds of SELEX and two rounds of counter-SELEX(against SELEX equipment and negative stool) were performed prior tosequencing for both NV and TV VPgs. Sequences obtained are in Tables 16and 17.

TABLE 15 Oligonucleotides Used for VPg Cloning SEQ NameSequence (5′-3′)* ID NO Norwalk VPg CCGGAATTCGGAAAGAACAAAGGCAAGACC 158Forward Norwalk VPg CCGCTCGAGTTCAAAATTGATCTTTTCATT 159 Reverse ATAATTulane VPg CCGGAATTCGCCAAGGGCAAGACAAAAAGG 160 Forward Tulane VPgCCGCTCGAGCTACTCGTCGTAATAATCATC 161 Reverse ACTGGG

TABLE 16 Representative Norwalk Virus (GI.1) VPg Aptamer CandidatesRound of SEQ # Aptamer SELEX Random Region Sequence* ID NO RepeatsIdentifier 8 CAGAGTTGATGTAAGCTTCGTGTTAGCTCAACTCTTATCG 66 10/19 N6 8AGGGATGTGTTGGATGCATGCCAGGCTTGGTAACATTGTA 67  1/19 N3 8TCTTCGGTTTAATAAAGTTGGCTAGGAAAGTTTAAAACCG 68  3/19 N1 8AGTGGGTGGTGATGAATTCTGGTCGCGCTGACAACCCGCG 69  1/19 N14 8CGGGTCTCGTCTATGCAGTACTCAAAACGCTTGAGGTACCGA 70  1/19 N1-2 8AAGGCTTTTTTAAAGGCTAGGCTTGATAATCGGTTAACTC 71  1/19 N4-2 8TGTCGATAAAGTGAGTTAAGTCACCGGCCCGGCCTATTCG 72  1/19 N11-2 8TCACACTCGTTTCTATTACTAAAACATCGTTCCTTTCAGC 73  1/19 N12-2 *Bolded sequencealso occurs in Tulane virus VPg aptamer pool

TABLE 17 Representative Tulane Virus VPg Aptamer Candidates Round of SEQ# Aptamer SELEX Random Region Sequence ID NO Repeats Identifier 8TCACACTCGTTTCTATTACTAAAACATCGTTCCTTTCAGC 74 13/17 T5 8TGGAAGGCGGGAAGATTTTTGGTCGACCTGACAACCCGGT 75  1/17 T9 8TAGTAACGATTACCAAAATTCTCCCGAGGCTGACAACCCG 76  1/17 T1-2 8TCGAGGTATGGCCTTGTCTAGGCGCACCTGACAACCCGGTG 77  1/17 T9-2 8TGTCGTTAATTATTCGTGATCTGACAACCCGATCACTCTC 78  1/17 T10-2 *Bolded sequencealso occurs in Norwalk virus VPg aptamer pool.

The VPg aptamers could be used to design a more stringent “integrated”in vitro process for discriminating infectious from non-infectiousvirus, especially if combined with an intact capsid recognition step(such as capture with an antibody or HBGA). The combination of these twosteps would allow the simultaneous confirmation of capsid and genomeintegrity. Since some disinfection methods degrade the capsid, andothers degrade the genome, this approach could also be used toinvestigate the mechanism of action of existing and new chemical orphysical disinfectants or sanitizers. For example, no one has studiedthe heat stability of VPg; theoretically the capsid, which is composedof quite stable dimers, may be more heat tolerant than the VPg (whichmust first be cleaved, and has unstructured regions flanking a core ofalpha helices as seen in FCV and MNV proteins using NMR).

In addition, the VPg aptamers could be a potentially more sensitiveclinical detection platform because a copy of the VPg is covalentlylinked to genomic and subgenomic RNA. Hence, there are more VPgs in eachvirus-infected cell than there are accessible binding sites for a ligandon a capsid. Moreover, because the VPg is under less selection pressurethan the capsid, the more conserved nature of it is likely to makeaptamers against it more cross-reactive and thus effective against awider range of HuNoV strains in clinical detection. Additionally, theseaptamers could be relevant in studying virus-infected cells as a cheaperalternative to VPg antibodies (which do not exist commerciallycurrently) for certain applications.

Similarly, the aptamers could be used as the recognition element in abiosensor that may be more sensitive than any other recognitionelements. Due to the ease with which they can be chemically modified,their high stability, and lower cost, such aptamers are more attractiveas candidates for ligands in biosensors, especially for point-of-careand in-field testing applications.

What is claimed is:
 1. A method of detecting the presence of at leastone norovirus strain in a test sample, the method comprising: (a)contacting said test sample with a norovirus-binding aptamer comprisinga norovirus-binding motif and/or any one of SEQ ID NOS: 1-78 and 176-181or variants thereof having at least 90% sequence identity and havingnorovirus-binding activity; and (b) detecting the presence of saidnorovirus-binding aptamer bound to norovirus in said test sample,wherein detection of bound aptamer indicates the presence of at leastone norovirus strain.